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 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.

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.

The Potential of Essential Oils from Active Packaging to Reduce Ethylene Biosynthesis in Plant Products. Part 1: Vegetables (Broccoli and Tomato).

Essential oils (EOs) extracted from plants have a high potential to reduce ethylene biosynthesis, although their effects have not been deeply studied yet on the key components of the ethylene biosynthesis pathway: l-aminocyclopropane-1-carboxylic (ACC) oxidase activity, ACC synthase activity, and ACC content. Hence, the present study aimed to elucidate the effects of released EOs from active packaging (with different EO doses ranging from 100 to 1000 mg m-2) on the ethylene biosynthesis key components of broccoli and tomato under different storage temperature scenarios. The largest ethylene inhibitory effects on broccoli and tomatoes were demonstrated by grapefruit EO and thyme essential EO (up to 63%), respectively, which were more pronounced at higher temperatures. Regarding EO doses, active packaging with a thyme EO dose of 1000 mg m-2 resulted in the strongest reduction (33-38%) of ethylene production in tomatoes. For broccoli, identical results were shown with a lower grapefruit EO dose of 500 mg m-2. The studied EO-active packaging decreased ACC synthase and ACC oxidase activities by 40-50% at 22 °C. Therefore, this EO-active packaging is a natural and effective technology to reduce ethylene biosynthesis in broccoli and tomatoes when they are stored, even in unsuitable scenarios at high temperatures.

The Potential of Essential Oils from Active Packaging to Reduce Ethylene Biosynthesis in Plant Products. Part 2: Fruits (Blueberries and Blackberries).

Plant essential oils (EOs) have an important ability to inhibit ethylene biosynthesis. Nevertheless, the effects of EOs on the key components of ethylene biosynthesis (l-aminocyclopropane-1-carboxylic (ACC) oxidase activity, ACC synthase activity, and ACC content) have not yet been thoroughly studied. Accordingly, this study focused on the effects of emitted EOs from active packaging (EO doses from 100 to 1000 mg m-2) on the key components of ethylene biosynthesis of blueberries and blackberries under several storage temperatures. Anise EO and lemon EO active packaging induced the greatest inhibitory effects (60-76%) on the ethylene production of blueberries and blackberries, respectively, even at high storage temperatures (22 °C). In terms of EO doses, active packaging with 1000 mg m-2 of anise EO or lemon EO led to the highest reduction of ethylene production, respectively. At 22 °C, the investigated EO active packing reduced the activities of ACC synthase and ACC oxidase up to 50%. In order to minimise ethylene biosynthesis in blueberries and blackberries when they are stored even under improper temperature scenarios at high temperatures, this EO active packaging is a natural and efficient technological solution.

Fruit ripening under heat stress: The intriguing role of ethylene-mediated signaling.

Crop production is significantly influenced by climate, and even minor climate changes can have a substantial impact on crop yields. Rising temperature due to climate change can lead to heat stress (HS) in plants, which not only hinders plant growth and development but also result in significant losses in crop yields. To cope with the different stresses including HS, plants have evolved a variety of adaptive mechanisms. In response to these stresses, phytohormones play a crucial role by generating endogenous signals that regulate the plant's defensive response. Among these, Ethylene (ET), a key phytohormone, stands out as a major regulator of stress responses in plants and regulates many plant traits, which are critical for crop productivity and nutritional quality. ET is also known as a ripening hormone for decades in climacteric fruit and many studies are available deciphering the function of different ET biosynthesis and signaling components in the ripening process. Recent studies suggest that HS significantly affects fruit quality traits and perturbs fruit ripening by altering the regulation of many ethylene biosynthesis and signaling genes resulting in substantial loss of fruit yield, quality, and postharvest stability. Despite the significant progress in this field in recent years the interplay between ET, ripening, and HS is elusive. In this review, we summarized the recent advances and current understanding of ET in regulating the ripening process under HS and explored their crosstalk at physiological and molecular levels to shed light on intricate relationships.

Targeted modification of CmACO1 by CRISPR/Cas9 extends the shelf-life of Cucumis melo var. reticulatus melon.

The gaseous plant hormone ethylene is a regulator of fruit shelf-life, one of the essential traits in fruits. Extending fruit shelf-life reduces food loss, thereby expected to contribute to food security. The enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) is the final step of the ethylene production pathway. Its suppression via antisense technology has been demonstrated to extend the shelf-life of melon, apple, and papaya. Genome editing technology is an innovative technique for plant breeding. Because the genome editing technology would not leave the exogenous genes in the final crop products, the crops via genome editing can be considered non-genetically modified yields; compared to conventional breeding, such as mutation breeding, the breeding term would be expected to be relatively short. These points include the advantage of this technique in utilization for commercial applications. We attempted to extend the shelf-life of the Japanese luxury melon (Cucumis melo var. reticulatus, 'Harukei-3') via modification of the ethylene synthesis pathway with the genome editing technology, CRISPR/Cas9 system. The Melonet-DB (https://melonet-db.dna.affrc.go.jp/ap/top) showed that the melon genome had the five CmACOs and the gene CmACO1 predominantly expressed in harvested fruits. From this information, CmACO1 was expected to be a key gene for shelf-life in melons. Based on this information, the CmACO1 was selected as the target of the CRISPR/Cas9 system and introduced the mutation. The final product of this melon did not have any exogenous genes. The mutation was inherited for at least two generations. In the T2 generation, the fruit phenotypes 14 days after harvest were as follows: ethylene production was reduced to one-tenth that of the wild type, pericarp colour remained green, and higher fruit firmness. Early fermentation of the fresh fruit was observed in the wild-type fruit but not in the mutant. These results show that CmACO1 knockout via CRISPR/Cas9 extended the melon's shelf-life. Moreover, our results suggest that genome editing technology would reduce food loss and contribute to food security.

[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.

Effect of ACC oxidase gene AhACOs on salt tolerance of peanut / 生物工程学报

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.

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.

A 1-aminocyclopropane-1-carboxylic-acid (ACC) dipeptide elicits ethylene responses through ACC-oxidase mediated substrate promiscuity.

Plants produce the volatile hormone ethylene to regulate many developmental processes and to deal with (a)biotic stressors. In seed plants, ethylene is synthesized from 1-aminocyclopropane-1-carboxylic acid (ACC) by the dedicated enzyme ACC oxidase (ACO). Ethylene biosynthesis is tightly regulated at the level of ACC through ACC synthesis, conjugation and transport. ACC is a non-proteinogenic amino acid, which also has signaling roles independent from ethylene. In this work, we investigated the biological function of an uncharacterized ACC dipeptide. The custom-synthesized di-ACC molecule can be taken up by Arabidopsis in a similar way as ACC, in part via Lysine Histidine Transporters (e.g., LHT1). Using Nano-Particle Assisted Laser Desoprtion/Ionization (Nano-PALDI) mass-spectrometry imaging, we revealed that externally fed di-ACC predominantly localizes to the vasculature tissue, despite it not being detectable in control hypocotyl segments. Once taken up, the ACC dimer can evoke a triple response phenotype in dark-grown seedlings, reminiscent of ethylene responses induced by ACC itself, albeit less efficiently compared to ACC. Di-ACC does not act via ACC-signaling, but operates via the known ethylene signaling pathway. In vitro ACO activity and molecular docking showed that di-ACC can be used as an alternative substrate by ACO to form ethylene. The promiscuous nature of ACO for the ACC dimer also explains the higher ethylene production rates observed in planta, although this reaction occurred less efficiently compared to ACC. Overall, the ACC dipeptide seems to be transported and converted into ethylene in a similar way as ACC, and is able to augment ethylene production levels and induce subsequent ethylene responses in Arabidopsis.

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.

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.

24-Epibrasinolide Modulates the Vase Life of Lisianthus Cut Flowers by Modulating ACC Oxidase Enzyme Activity and Physiological Responses.

Ethylene is the most important factor playing roles in senescence and deterioration of harvested crops including cut flowers. Brassinosteroids (BRs), as natural phytohormones, have been reported to differently modulate ethylene production and related senescence processes in different crops. This study was carried out to determine the effects of different levels of 24-epibrassinolide (EBL) on ACC oxidase enzyme activity, the final enzyme in ethylene biosynthesis pathway, vase life, and senescence rate in lisianthus cut flowers. Harvested flowers were treated with EBL (at 0, 3, 6, and 9 µmol/L) and kept at 25 °C for 15 days. The ACC oxidase activity, water absorption, malondialdehyde (MDA) production and vase solution absorption rates, chlorophyll and anthocyanin contents, and the vase life of the flowers were evaluated during and at the end of storage. EBL at 3 µmol/L significantly (p ≤ 0.01) enhanced the flower vase life by decreasing the ACC oxidase activity, MDA production and senescence rates, and enhancing chlorophyll and anthocyanin biosynthesis and accumulation, relative water content, and vase solution absorption rates. By increasing the concentration, EBL negatively affected the flower vase life and postharvest quality probably via enhancing the ACC oxidase enzyme activity and subsequent ethylene production. EBL at 6 and 9 µmol/L and in a concentration dependent manner, enhanced the ACC oxidase activity and MDA production rate and decreased chlorophyll and anthocyanin accumulation and water absorption rate. The results indicate that the effects of brassinosteroids on ethylene production and physiology of lisianthus cut flowers is highly dose dependent.

Effects of plant growth-promoting rhizobacteria strains producing ACC deaminase on photosynthesis, isoprene emission, ethylene formation and growth of Mucuna pruriens (L.) DC. in response to water deficit.

Agricultural sustainability is an increasing need considering the challenges posed by climate change and rapid human population growth. The use of plant growth-promoting rhizobacteria (PGPR) may represent an excellent, new agriculture practice to improve soil quality while promoting growth and yield of important crop species subjected to water stress conditions. In this study, two PGPR strains with 1-Aminocyclopropane-1-Carboxylate (ACC) deaminase activity were co-inoculated in velvet bean plants to verify the physiological, biochemical and molecular responses to progressive water stress. The results of our study show that the total biomass and the water use efficiency of inoculated plants were higher than uninoculated plants at the end of the water stress period. These positive effects may be derived from a lower root ACC content (-45 %) in water-stressed inoculated plants than in uninoculated ones resulting in lower root ethylene emission. Furthermore, the ability of inoculated plants to maintain higher levels of both isoprene emission, a priming compound that may help to protect leaves from oxidative damage, and carbon assimilation during water stress progression may indicate the underlining metabolic processes conferring water stress tolerance. Overall, the experimental results show that co-inoculation with ACC deaminase PGPR positively affects tolerance to water deficit, confirming the potential for biotechnological applications in water-stressed agricultural areas.

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.

BES1 negatively regulates the expression of ACC oxidase 2 to control the endogenous level of ethylene in Arabidopsis thaliana.

Quantitative reverse transcription PCR (qRT-PCR) analysis and ProACO2::GUS expression showed that ACO2 was highly expressed in the shoots of Arabidopsis seedlings under light conditions. Exogenously applied aminocyclopropane-1-carboxylic acid (ACC) enhanced the expression of ACO2, whereas Co2+ ions suppressed its expression. In comparison with wild-type seedlings, the ACO2 knockdown mutant aco2-1 produced less ethylene, which resulted in the inhibited growth of Arabidopsis seedlings. Exogenously applied brassinolide reduced the expression of ACO2. ACO2 expression was increased in det2, a brassinosteroid (BR)-deficient mutant; however, it was decreased in bes1-D, a brassinosteroid insensitive 1-EMS-suppressor 1 (BES1)-dominant mutant. In the putative promoter region of ACO2, 11 E-box sequences for BES1 binding but not BR regulatory element sequences for brassinazole-resistant 1 (BZR1) binding were found. Chromatin immunoprecipitation assay showed that BES1 could directly bind to the E-boxes located in the putative promoter region of ACO4. Less ethylene was produced in bes1-D seedlings compared with wild-type seedlings, suggesting that the direct binding of BES1 to the ACO2 promoter may negatively regulate ACO2 expression to control the endogenous level of ethylene in Arabidopsis seedlings.

Effect of modified and controlled atmosphere storage on enzyme activity and senescence of Dendrobium orchids.

This research investigated the effect of different atmosphere storage conditions on 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase enzyme activity; and senescence of cut Dendrobium orchid flowers. The atmosphere storage conditions under study were normal atmosphere, modified atmosphere packaging, and controlled atmosphere. Under the normal atmosphere, carbon dioxide (CO2) and oxygen (O2) concentrations were 0.03 and 21 %, respectively. For the modified atmosphere packaging, cut orchid flowers were wrapped in polypropylene film prior to filling with 5 % CO2 and 2 % O2, while under the controlled atmosphere, CO2 and O2 concentrations were maintained at 5 and 2 %, respectively. The storage temperature and relative humidity were 13 °C and 95 %, respectively. The ACC synthase and ACC oxidase activity and ethylene-induced electrolyte leakage were determined and results compared. The controlled atmosphere substantially lowered ACC synthase and ACC oxidase activity and was effective in delaying senescence of cut orchid flowers, as indicated by the longest storage life of 28.33 days, followed by the modified atmosphere packaging (18.15 days) and normal atmosphere (11.67 days). The longer storage life enables suppliers of orchid flowers to efficiently manage the demand and supply and also provides exporters with new opportunities to expand into distant overseas markets. The novelty of this research lies in the use of different storage environments to investigate the senescence mechanisms at tissue level of Dendrobium orchid flowers in response to ACC synthase and ACC oxidase enzyme activity.

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.

Expression Fluctuations of Genes Involved in Carbohydrate Metabolism Affected by Alterations of Ethylene Biosynthesis Associated with Ripening in Banana Fruit.

The banana is a typical climacteric fruit that undergoes ethylene dependent ripening. During fruit ripening, ethylene production triggers a developmental cascade that results in a series of physiological and biochemical changes. The fruit transcriptomes of untransformated wild-type (WT) and RNAi transgenic banana plants for Mh-ACO1 and Mh-ACO2 have been previously sequenced and analyzed, and most of the differentially expressed genes were enriched in 'carbon fixation in photosynthetic organism', 'cysteine and methionine metabolism', 'citrate cycle (tricarboxylic acid cycle, TCA cycle)', and 'starch and sucrose metabolism' based on Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. In this research, we investigated the expression fluctuations of genes involved in carbohydrate metabolism affected by alterations of ethylene biosynthesis associated with ripening in banana fruits. Expression profiles of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase/ß-fructofuranosidase, as analyzed by Avadis and Trinity, showed that both analyses were complementary and consistent. The overall gene expression tendency was confirmed by the implementation of quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) with mRNAs of banana fruits in Mh-ACO1 and Mh-ACO2 RNAi transgenic plants. These results indicated that altered expression of genes associated with ethylene biosynthesis strongly influenced the expression levels of genes related to starch and sucrose metabolism, as well as the glycolysis pathway in ripening banana fruits.