ACC Synthesis Research Articles

A UPLC-MS/MS method for quantification of metabolites in the ethylene biosynthesis pathway and its biological validation in Arabidopsis.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

The miR156/SPL12 module orchestrates fruit colour change through directly regulating ethylene production pathway in blueberry.

Colour change is an important event during fruit ripening in blueberry. It is well known that miR156/SPLs act as regulatory modules mediating anthocyanin biosynthesis and ethylene plays critical roles during colour change, but the intrinsic connections between the two pathways remain poorly understood. Previously, we demonstrated that blueberry VcMIR156a/VcSPL12 affects the accumulation of anthocyanins and chlorophylls in tomato and Arabidopsis. In this study, we first showed that VcMIR156a overexpression in blueberry led to enhanced anthocyanin biosynthesis, decreased chlorophyll accumulation, and, intriguingly, concomitant elevation in the expression of ethylene biosynthesis genes and the level of the ethylene precursor ACC. Conversely, VcSPL12 enhanced chlorophyll accumulation and suppressed anthocyanin biosynthesis and ACC synthesis in fruits. Moreover, the treatment with ethylene substitutes and inhibitors attenuated the effects of VcMIR156a and VcSPL12 on pigment accumulation. Protein-DNA interaction assays indicated that VcSPL12 could specifically bind to the promoters and inhibit the activities of the ethylene biosynthetic genes VcACS1 and VcACO6. Collectively, our results show that VcMIR156a/VcSPL12 alters ethylene production through targeting VcACS1 and VcACO6, therefore governing fruit colour change. Additionally, VcSPL12 may directly interact with the promoter region of the chlorophyll biosynthetic gene VcDVR, thereby activating its expression. These findings established an intrinsic connection between the miR156/SPL regulatory module and ethylene pathway.

Differences in the Mode of Action of Florpyrauxifen-Benzyl between Barnyardgrass and Yerbadetajo

Barnyardgrass (a monocotyledon) and yerbadetajo (a dicotyledon) are the most troublesome weeds in rice fields in China. The synthetic auxin herbicide florpyrauxifen-benzyl can effectively control both weeds. The objective of this research was to clarify modes of action of florpyrauxifen-benzyl in barnyardgrass and yerbadetajo. Our results showed that yerbadetajo was more sensitive to florpyrauxifen-benzyl than barnyardgrass: the 50% growth rate inhibition in barnyardgrass and yerbadetajo was 4.14 and 0.38 g a.i. ha−1, respectively. Florpyrauxifen-benzyl induced the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and abscisic acid (ABA) in yerbadetajo within 24 h, while in barnyardgrass, the highest production occurred at 12 h and decreased at 24 h. ACC and ABA levels in yerbadetajo at 24 h of treatment were significantly higher than those in barnyardgrass at any time. There were less differentially expressed genes related to ethylene and ABA synthesis in barnyardgrass than in yerbadetajo. There were five genes induced to increase by florpyrauxifen-benzyl in barnyardgrass and eleven genes in yerbadetajo. More ACC oxidase genes (ACO) were induced in barnyardgrass and more ACC synthesis genes (ACS) in yerbadetajo, especially three ACS3 (>30 fold). We speculated that differences in gene expression caused differences in ethylene and ABA production, leading to differences in phytotoxicity.

Growth Promotion of Guava “Pear” (Psidium guajava cv.) by Sinorhizobium mexicanum in Southern Mexican Agricultural Fields

Biofertilizers formulated with nitrogen-fixing bacteria represent an alternative to chemical fertilizers because they increase soil fertility and protect the environment. Therefore, the objective of this study was to analyze the effects on the growth of guava “pear” (Psidium guajava cv.) after inoculation with a nitrogen fixing bacterium Sinorhizobium mexicanum ITTG-R7T. The study was carried out in an agricultural rural area of Chiapas, Mexico, where farmers do not have programs of regenerative agriculture. First, the agricultural soil was subjected to physicochemical and metagenomic analysis in order to determine the soil quality and its bacterial community composition. Likewise, multifunctional biochemical tests and plant inoculation assays were evaluated to determine the potential of S. mexicanum ITTG-R7T as plant-growth-promoting bacteria (PGPB). The site was rain fed and had silty clay loam soil with abundant Bacillaceae. S. mexicanum ITTG-R7T showed different properties as PGPB such as the production of indole compounds, synthesis of extracellular enzymes, phosphate solubilization, synthesis of siderophores, ACC (1-aminocyclopropane-1-carboxylate) deaminase, and nitrogenase activity (ARA). When the strain ITTG-R7 T was combined with chemical nutrients, it had the highest positive effect on the growth and development of guava plants. Guava biofertilization with ITTG-R7T had a significant influence (p < 0.05) mainly on the total plant height (368.83 cm), number of flowers (36.0) and the amount of chlorophyll (2.81 mg mL−1) in comparison with the other treatments evaluated. ITTG-R7T is a promising strain for improving the guava crop yield.

α-aminoisobutyric acid mimics the effect of 1-aminocyclopropane-1-carboxylic acid to promote sexual reproduction in the marine red alga Pyropia yezoensis (Rhodophyta)

1-aminocyclopropane-1-carboxylic acid (ACC), a precursor for ethylene, stimulates the switch from a vegetative to a sexual reproductive phase in the marine red alga Pyropia species. This study explored the effects of ethylene biosynthesis inhibitors on the sexual reproduction of gametophytes of the red alga Pyropia yezoensis to gain a functional understanding of the role of ACC as a plant hormone in red algae. Here we show that two inhibitors of ACC synthesis in higher plants, 2-aminoethoxyvinyl glycine (AVG) and aminooxyacetic acid (AOA), had no effect on the growth and gametogenesis of P. yezoensis. In contrast, exogenous application of α-aminoisobutyric acid (AIB), a structural analog of ACC that blocks the conversion of ACC to ethylene in higher plants, induced the formation of spermatangia and carpospores in a similar manner as ACC, without endogenous ACC accumulation. The treatment of AIB failed to inhibit ethylene production in the gametophytes. The present results suggest that AIB mimics the effect of ACC to induce the sexual reproduction and support our previous study that ACC has a role in the regulation of the sexual reproduction independent from ethylene.

Identifying the Pressure Points of Acute Cadmium Stress Prior to Acclimation in Arabidopsis thaliana.

The toxic metal cadmium (Cd) is a major soil pollutant. Knowledge on the acute Cd-induced stress response is required to better understand the triggers and sequence of events that precede plant acclimation. Therefore, we aimed to identify the pressure points of Cd stress using a short-term exposure set-up ranging from 0 h to 24 h. Acute responses related to glutathione (GSH), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and the oxidative challenge were studied at metabolite and/or transcript level in roots and leaves of Arabidopsis thaliana either exposed or not to 5 µM Cd. Cadmium rapidly induced root GSH depletion, which might serve as an alert response and modulator of H2O2 signalling. Concomitantly, a stimulation of root ACC levels was observed. Leaf responses were delayed and did not involve GSH depletion. After 24 h, a defined oxidative challenge became apparent, which was most pronounced in the leaves and concerted with a strong induction of leaf ACC synthesis. We suggest that root GSH depletion is required for a proper alert response rather than being a merely adverse effect. Furthermore, we propose that roots serve as command centre via a.o. root-derived ACC/ethylene to engage the leaves in a proper stress response.

Melatonin enhances salt tolerance by promoting MYB108A-mediated ethylene biosynthesis in grapevines

The signal molecules melatonin and ethylene play key roles in abiotic stress tolerance. The interplay between melatonin and ethylene in regulating salt tolerance and the underlying molecular mechanism of this interplay remain unclear. Here, we found that both melatonin and 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene) enhanced the tolerance of grapevine to NaCl; additionally, ethylene participated in melatonin-induced salt tolerance. Further experiments indicated that exogenous treatment and endogenous induction of melatonin increased the ACC content and ethylene production in grapevine and tobacco plants, respectively. The expression of MYB108A and ACS1, which function as a transcription factor and a key gene involved in ethylene production, respectively, was strongly induced by melatonin treatment. Additionally, MYB108A directly bound to the promoter of ACS1 and activated its transcription. MYB108A expression promoted ACC synthesis and ethylene production by activating ACS1 expression in response to melatonin treatment. The suppression of MYB108A expression partially limited the effect of melatonin on the induction of ethylene production and reduced melatonin-induced salt tolerance. Collectively, melatonin promotes ethylene biosynthesis and salt tolerance through the regulation of ACS1 by MYB108A.

Aminooxyacetic acid (АОА), inhibitor of 1-aminocyclopropane-1-carboxilic acid (AСС) synthesis, suppresses self-incompatibility-induced programmed cell death in self-incompatible Petunia hybrida L. pollen tubes.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.

Isolation and characterization of S-Adenosylmethionine synthase gene from cucumber and responsive to abiotic stress

S-adenosylmethionine synthetase (SAMS) catalyzes methionine and ATP to generate S-adenosyl-L-methionine (SAM). In plants, accumulating SAMS genes have been characterized and the majority of them are reported to participate in development and stress response. In this study, two putative SAMS genes (CsSAMS1 and CsSAMS2) were identified in cucumber (Cucumis Sativus L.). They displayed 95% similarity and had a high identity with their homologous of Arabidopsis thaliana and Nicotiana tabacum. The qRT-PCR test showed that CsSAMS1 was predominantly expressed in stem, male flower, and young fruit, whereas CsSAMS2 was preferentially accumulated in stem and female flower. And they displayed differential expression profiles under stimuli, including NaCl, ABA, SA, MeJA, drought and low temperature. To elucidate the function of cucumber SAMS, the full-length CDS of CsSAMS1 was cloned, and prokaryotic expression system and transgenic materials were constructed. Expressing CsSAMS1 in Escherichia coli BL21 (DE3) improved the growth of the engineered strain under salt stress. Overexpression of CsSAMS1 significantly increased MDA content, H2O2 content, and POD activity in transgenic lines under non-stress condition. Under salt stress, however, the MDA content of transgenic lines was lower than that of the wild type, the H2O2 content remained high, the polyamine and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way. Taken together, our results suggested that both CsSAMS1 and CsSAMS2 were involved in plant development and stress response, and a proper increase of expression level of CsSAMS1 in plants is benificial to improving salt tolerance.

Effects of alternate bearing and 2,4-D application on fruit growth pattern, abscission enzymes activity, ACC content of calyx and carbohydrates partitioning of fruits in Satsuma mandarin ( Citrus unshiu Marc.) cv. Miyagawa

Abstract Considerable fluctuations in annual yield (alternate bearing) is one of the noticeable characteristics of some citrus cultivars. Application of chemical thinners at high yield (On) years is an efficient way for ameliorating this problem. The aim of this work was to evaluate the effects of crop load and 2,4-D foliar application at full bloom on Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa fruits at On year under rainfed farming. The 24-year-old Miyagawa mandarin trees grafted on Poncirus (Poncirus trifoliata Raf.) rootstock grown at North Iran were examined for ethylene precursor (ACC) content of calyx, activity of cellulase and polygalacturonase in abscission zone of fruitlets, and also the growth pattern and carbohydrates partitioning in fruits. Results showed that 2,4-D was effective in intensifying the activity of abscission enzymes and ACC synthesis during fruitlet abscission and increased fruitlets drop. In addition, 2,4-D significantly increased fruit growth rate from 73 days after anthesis (DAA), resulting to significantly heavier fruits than those of On (check) trees. The other consequence of 2,4-D treatment was the increase in the fruits’ sucrose, fructose, and glucose levels, resulting in fruit quality improvement. As a result, 2,4-D spray of On Miyagawa trees during full bloom period can be a suitable solution to control alternate bearing and to enhance fruit quality.

Role of abscisic acid and ethylene in the control of water transport-driving forces in germinating petunia male gametophyte

Data on ABA involvement in osmoregulation of in vitro germinating petunia (Petunia hybrida L.) male gametophyte were obtained. Two potential targets of ABA action in a pollen tube (PT) are identified. These are represented by (1) plasma membrane (PM) H+-ATPase, electrogenic proton pump participating in PM polarization, and (2) Ca-dependent K+-channels localized in the same membrane. It was established that a stimulatory effect of ABA on electrogenic activity of H+-ATPase is mediated by the increase in free Ca2+ level in the cytosol of a PT and reactive oxygen species (ROS) generation. Based on the results obtained on the role of K+ ions in the hormonal control of water transport-driving forces in a PT, the hypothesis suggesting that ABA stimulated pollen grains (PGs) germinating and PT growth by activating K+-channels was put forward. The revealed ABA-induced shift in cytoplasmic pH (pHc) is suggested to be involved in a cascade of the events of the progamic phase of fertilization, including pH-dependent K+-channels functioning. It was established that ABA abolishes the inhibitory effects of ethylene receptors blocker, 1-methylcyclopropene (1-MCP), and blockers of ACC and ABA synthesis (aminooxyacetic acid, AOA, and fluridone, respectively) on PT germination and growth, whereas ethrel blocks the inhibitory effect of fluridone on PT growth. In stigmas pretreated with ABA and AOA before pollination, this phytohormone was found to suppress inhibitory effect of AOA on ACC synthesis in the pollen-pistil system. All these findings, taken together, led us to the conclusion that ABA is involved in petunia male gametophyte osmoregulation interacting with ethylene at the level of ACC synthesis in the progamic phase of fertilization.

Auxin abolishes inhibitory effects of methylcyclopropen and amino oxyacetic acid on pollen grain germination, pollen tube growth, and the synthesis of ACC in petunia

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.

Physiological responses of the co-cultivation of PGPR with two wheat cultivars in vitro under stress conditions

Wheat (Triticum aestivum L.) may be exposed to different stress conditions that influence its productivity. In southern Brazil the rainfed wheat crop is limited by dry spells consisting of short periods of drought during rainy periods. One way to confer tolerance to the drought´s effects and stimulate plant productivity is the action of a group of bacteria capable of making association with plants, known as Plant Growth Promoter Bacteria (PGPB), as Herbaspirillum seropedicae and Azospirillum brasilense, both diazotrophic. It was demostrated that the PGPB can benefit the plants in several ways: synthesizing some phytohormones, siderophores, biological nitrogen fixation, inducing systemic resistance etc. The strain H. seropedicae SmR1 unlike A. brasilense AbV5, presents a gene encoding the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks ACC, the ethylene precursor in alpha-keto-butyric acid (AKB) and ammonium ion. The indole-3-acetic acid (IAA) is a phytohormone, produced by PGPB that modulates the synthesis of plant ethylene, and it is known as stress and senescence promoter. The IAA can stimulate the synthesis of ACC by increasing the activity of ACC synthase and inhibit the transformation of ACC into ethylene by decreasing the activity of ACC oxidase. The aim of this work was study the in vitro interactions of two varieties of wheat, CD120 and an ancestor cultivar Frontana, with the H. seropedicae SmR1 and A. brasilense AbV5 to verify the physiological changes in the culture medium under osmotic stress imposed by polyethylene glycol (PEG 6000). Wheat plantlets were obtained throughout mature embryos cultured on solid MS medium, aseptically transferred after 28 days to 10 mL of 1/10 MS liquid medium with or without PEG and inoculated with H. seropedicae and/or A. brasilense. The controls consisted of wheat plantlets with and without PEG in the same condition. After 5 days, plantlets and bacteria were harvested and the co-culture medium was taken to determine the concentrations of IAA (Glickmann & Dessaux, 1995) [1], AKB (Penrose and Glick, 2003) [2], and total protein (Hartree, 1972) [3]. The treatments of CD120 without PEG and with H. seropedicae showed the highest IAA concentration. In Frontana the amounts of IAA are lower as compared to CD120, however, treatments under the stress condition (with PEG) and A. brasilense stood out. Treatments with PEG and both bacteria showed similar performances associated with CD120. The amounts of AKB increased in the presence of PEG and H. seropedicae for both cultivars, probably by action of ACC deaminase activity. The total protein increased in the presence of PEG comparing the two controls for both cultivars. The total protein in CD120 was greater only when bacteria were present compared to their controls indicating a plant cell death for CD120 and Frontana. It is possible to conclude that the association of H. seropedicae is more effective with CD120 and indicated the reduction of ethylene. Frontana associates better with A. brasilense Possibly PEG causes cell death in the system or exudation of proteins by the cultivars tested. These data corroborate with the literature where plant vs. bacteria interaction is genotype and strain dependent.

Ethylene Biosynthetic Responses of Mango (Mangifera indica L. cv. ‘Carabao’) Fruits to 1-methylcyclopropene (1-MCP) Preharvest Aqueous Spray Applications

800x600 The ethylene antagonist 1-methylcyclopropene (1-MCP) was used as a tool to investigate the mechanism of ethylene feedback regulation in mango (Mangifera indica L. cv. ‘Carabao’) fruits. The ethylene biosynthetic responses of mango fruits to 1-MCP applied prior the upsurge in ethylene production on-tree which was around 100 days after flower induction (DAFI) were studied. Aqueous 1-MCP spray formulation with 10 ppm concentration was applied to on-tree mango fruits at 100 DAFI. From these sprayed fruits, four (4) sets of fruits were tagged for a second round of 10 ppm aqueous 1-MCP spraying. One set was sprayed the second time at 105 DAFI, while the remaining sets at 110, 115 and 120 DAFI respectively. The positive control was sprayed with 0 ppm 1-MCP while the negative controls were the unsprayed fruits. Representative fruit samples from each set were harvested 2 d after spraying and every 2 d thereafter until reaching 120 DAFI. Right after each harvest, samples were analyzed for internal ethylene, ethylene production, ACC levels and ACC oxidase activities. The negative control exhibited two internal ethylene peaks before harvest maturity. However, in 1-MCP treated fruits, low levels of ethylene were detected and the second peak was not observed. A single application of 1-MCP at 100 DAFI resulted in lower ethylene production compared with controls. However, a second application proved to be more effective as the lowering of ethylene production was carried over until the day of harvest. The levels of 1-aminocyclopropane-1-carboxylate (ACC) increased after 110 DAFI for the controls. Reapplication of 1-MCP at 110 DAFI resulted to lower ACC levels, which was not observed with the other treatments with 1-MCP reapplication. Possibly, new ethylene receptors are synthesized at 110 DAFI, and 1-MCP reapplication at this time effectively blocks ethylene binding to the new receptors. ACC oxidase activity was lowered by 1-MCP applied at 100 DAFI compared with the controls. However, trends in ACC oxidase activities were almost the same for all the treatments and controls after 105 DAFI. Thus, ACC oxidase proceeds with its normal activity without being inhibited by 1-MCP. This suggests that ethylene biosynthesis in ‘Carabao’ mango fruit is regulated at the level of ACC synthase and not ACC oxidase. Thus, autocatalytic ethylene biosynthesis in ‘Carabao’ mango can be controlled by regulating ACC synthase activity, which proves that ACC synthesis is the rate limiting step in ethylene feedback mechanism in ‘Carabao’ mango. Normal 0 false false false EN-PH X-NONE X-NONE

Dietary Milk Polar Lipids Benefit Gut Barrier Integrity And Lipid Metabolism In C57BL/6J Mice During Systemic Inflammation Induced By Escherichia Coli Lipopolysaccharide

To explore dynamic interactions among dietary fat, gut permeability and lipid metabolism in the context of systemic inflammation, 3 groups of C57BL/6J mice were fed for 8 weeks: (1) modified AIN‐93G diet (CO); (2) CO with milk gangliosides (GG); (3) CO with milk phospholipids (PL). After 2 weeks’ feeding, animals were injected subcutaneously with Escherichia coli lipopolysaccharide (LPS) endotoxin to induce acute inflammation. Animals were fed for 6 more weeks to observe recovery. Gut permeability was assessed by differential sugar tests at baseline and 24h, 2 week plus 4 week after LPS stress. GG protected intestinal integrity against LPS stress while PL facilitated the recovery of small intestine barrier function. Both GG and PL suppressed colon permeability increase against LPS stress and facilitated barrier integrity recovery while PL had a bigger effect for prevention and GG had a stronger effect for recovery. GG or PL did not affect food intake, weight gain or fasting plasma levels of glucose, cholesterol and triglycerides. Expressions of 13 genes on liver lipid metabolism were assessed by RT‐qPCR. GG increased beta‐oxidation gene ACAA compared with CO & PL. PL suppressed fatty acid synthesis gene ACC and cholesterol synthesis gene HMGCR compared with CO & GG. In general, dietary milk polar lipids may have beneficial effects on gut permeability and lipid metabolism in the context of systemic inflammation.Grant Funding Source: Western Dairy Center

The nonclimacteric guava cultivar ‘Jen-Ju Bar’ is defective in system 2 1-aminocyclopropane-1-carboxylate synthase activity

The ripening characteristics and physiology of two guava cultivars, ‘Jen-Ju Bar’ (‘JJB’) and ‘Li-Tzy Bar’ (‘LTB’), were investigated and compared because of their divergent storability. During storage at 20°C, mature-green ‘LTB’ fruit exhibited ripening behavior typical for a climacteric fruit, including increases in respiration and ethylene production (3.75mmolCO2kg−1h−1 and 1.37×103nmolC2H4kg−1h−1 at peak levels, respectively), yellow color development, flesh softening, and volatile production. By contrast, ‘JJB’ is considered a nonclimacteric fruit because it features low and stable rates of respiration (0.61–0.73mmolCO2kg−1h−1) and ethylene production (less than 4nmolC2H4kg−1h−1) while retaining its pale-green color and firmness throughout a 20d observation period. Furthermore, the nonclimacteric nature of ‘JJB’ fruit was revealed through its responses to various levels of applied ethylene, as its respiration rate increased in a concentration-dependent manner. This respiration enhancement occurred only when exogenous ethylene was present and could be evoked repeatedly in the same ‘JJB’ fruit. The ripening characteristics of ‘JJB’ do not result from a defect in ethylene signaling, but instead result from a lack of autocatalytic ethylene synthesis, as propylene ventilation stimulated normal fruit ripening and an upsurge of respiration without a massive endogenous ethylene release. However, a cutting treatment of ‘JJB’ was able to induce stress-responsive ethylene production expected from system 1 ethylene biosynthesis. The finding that 1-aminocyclopropane-1-carboxylic acid (ACC) content and ACC synthase (ACS) activity were significantly lower in ‘JJB’ than in ‘LTB’ fruit after harvest, together with the finding that treatment with ACC (rather than S-adenosyl-l-methionine) led to both fully ripened ‘JJB’ fruit and endogenous ethylene production, strongly suggest that insufficient ACC synthesis, i.e., the reaction catalyzed by ACS, prevents system 2 ethylene biosynthesis in ‘JJB’ guava fruit.

Effect of Exogenous Ethylene on ACC Content and ACC Oxidase Activity During Ripening of Manila Mangoes Subjected to Hot Water Treatment

Mangoes (Mangifera indica L.) 'Manila' were subjected to the USDA-approved hot water treatment and then exposed to synthetic air mixtures containing 0.5, 0.75 or 1 ml l(-1) of ethylene for 6, 12 or 18 h at 25 degrees C, to induce accelerated ripening. After treatment the mangoes were allowed to ripen in air at 24-25 degrees C. The content of 1-aminocyclopropane-1-carboxylic acid (ACC) and ACC oxidase (ACO) activity increased in fruit treated with 0.5 and 0.75 ml l(-1) of ethylene for 6 or 12 h. Ethylene production was reduced in fruit treated with 1 ml l(-1) of ethylene. This was due to the decreased of ACC synthesis rather than to lower ACC oxidase activity. Treatment with 0.5 ml l(-1) of ethylene for 12 h was found best for accelerate ripening; fruits were fully ripened and edible 3 days after treatment, compared to 6-7 days for untreated mangoes.

Relationships of Ethylene Evolution Rate and 1-Aminocylopropane -1-Carboxylic Acid Concentration in Grains during Filling Period with Appearance Quality of Rice

To elucidate the relationship between ethylene evolution from the grains and the appearance quality of rice, ten different rice genotypes were used to determine the ethylene evolution rate, 1-aminocylopropane-1-carboxylic acid (ACC) concentration in grains during grain filling and the appearance quality of rice, and the effects of chemical regulators on concentrations of ethylene and ACC in the grains during grain filling were also investigated to verify the roles of ethylene in the rice quality formation. The ethylene evolution rates and ACC concentrations in grains during the mid and late grain filling stages were very significantly and positively correlated with chalky kernel percentage and chalkiness. The cultivars with a low ACC concentration in grains exhibited a close amyloplast arrangement and small space between starch granules, whereas those with a high ACC concentration in grains showed a loose arrangement and wide space between the granules. Application of 1 μmol/L ACC to panicles at mid and late grain filling stages significantly loosened amyloplast arrangement and increased chalky kernel percentage, chalky area and chalkiness, and the results were reversed when 1 μmol/L amino-ethoxyvinylglycine, an inhibitor of ACC synthesis enzyme, was applied to panicles. A practice of moderate dry-wet alternate irrigation reduced ethylene evolution and ACC concentration in grains and thereby reduced chalkiness. The results suggested that ethylene and ACC in grains play an important role in the endosperm structure and appearance quality of rice, and the appearance quality would be improved by reducing ethylene evolution and ACC in grains through either variety breeding and selection, or chemical regulations or cultivation techniques.

Determination of the Substrate Specificity of Tripeptidyl-peptidase I Using Combinatorial Peptide Libraries and Development of Improved Fluorogenic Substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.

Mechanism of resistance to quinclorac in smooth crabgrass ( Digitaria ischaemum)

The mechanism of resistance to quinclorac was investigated in a smooth crabgrass biotype [ Digitaria ischaemum (Schreb. ex Schweig) Schreb. ex Muhl] from Tulare County, California. Quinclorac (8.96 kg a.i. ha −1) had no effect ( P = 0.18) on the resistant (R) biotype, but reduced fresh weight of a susceptible (S) biotype by 93%. After treatment with 4.48 kg a.i. quinclorac ha −1, the S biotype produced about three times more ethylene than the R biotype and accumulated cyanide in tissues. Similar amounts of endogenous cyanide resulting from treatment with KCN reproduced quinclorac phytotoxicity. Pre-treatment with the ACC synthase inhibitor AVG reduced quinclorac phytotoxicity by 37% and ethylene production by 89%. These data suggest a target site-based mechanism of resistance involving stimulation of ACC synthesis and accumulation of cyanide. Also, the R biotype had four times more β-cyanoalanine synthase activity than the S biotype, suggesting a higher ability to detoxify cyanide.