Ethylene Inhibition and Control

//0 ADD SLIDE Ethylene Inhibition and Control Marita Cantwell Dept. Plant Sciences, UC Davis micantwell@ucdavis.edu Fruit Ripening and Retail Handling Workshop Postharvest Technology Center, UC Davis, March -, 0 Impact of CO on ethylene responses VENTILATION IS MORE COST EFFECTIVE THAN SCRUBBING BEST AIR FIRST TO LEAFY GREENS, THEN TO INTERMEDIATE TEMP ROOM AND AREAS AND THEN TO THE RIPENING ROOM AREA, SUCTION FAN IN RIPENING AREA FOR EXHAUST AND INCOMING AIR IN THE ETHYLENE SENSITIVE AREA Thanks to Michael Reid, UC Davis for many slides Some References Ethylene effects. M.E. Saltveit. 00. USDA handbook. Commercial Storage of Fruits, Vegetables http://www.ba.ars.usda.gov/hb/contents.html Recent advances in Ethylene Research. Z.F. Lin, S. Zhong and D. Grierson. 009. J. Expt. Bot. 0: -. Postharvest Technology Center; postharvest libraries: ethylene and -MCP; http://postharvest.ucdavis.edu/ The use of -Methylcyclopropene (-MCP) on fruits and vegetables. C. Watkins. 00. Biotech. Adv. : 9-09. The Dual Role of Ethylene in Postharvest Handling Useful: Accelerates ripening Chlorophyll degradation Causes abscission Problematic: Accelerates ripening Accelerates senescence and chlorophyll degradation Causes abscission Other undesirable changes Ethylene Production Rates by Fresh Produce at 0 C ( F) Range (µl/kg-h) Product 0.0-0. Citrus, grape, cherry strawberry MOST VEGETABLES 0.-.0 Pineapple, blueberry, cucumber.0-0.0 Banana, mango, tomato, honeydew melon, fig 0-00 Apple, avocado, cantaloupe, nectarine, papaya, pear >00 Cherimoya, passion fruit, sapotes Ethylene production and sensitivity information for specific products: Produce Facts: http://postharvest.ucdavis.edu/pf/ USDA Handbook : http://www.ba.ars.usda.gov/hb/ ppm 0.ppm 0.ppm 0ppm control Abscission of snapdragon flowers in response to ethylene shows a typical threshold and plateau response; cultivars to differ in threshold response Similar threshold for ethylene effects on lettuce and carrots From M. Reid, UC Davis

//0 Postharvest Compatibility Issues Temperature Relative Humidity Ethylene Odor Transportation and Loading Distribution warehouses and Storage rooms Ethylene can be detrimental for Fruits Fruit Kiwifruit Avocado, Fuyu Persimmon Citrus Stone fruits Watermelon Symptoms Softening; as little as 0 ppm induces softening Exposure to ppm at C increases chilling injury symptoms Use of ethylene for de-greening may increase senescence of peel and button Increase in decay associated with accelerated softening Tissue softening and breakdown Retail & Food Service outlets Detrimental Ethylene Effects on Vegetables The lower the temperature, less ethylene is produced by fruits At lower temperatures, vegetables are less affected (exceptions: lettuce, carrots) Problematic: Accelerates ripening Accelerates senescence Causes abscission Bitterness in carrots Manage Ethylene Effects. Temperature and Time. Avoidance. Removal. Inhibition of production. Inhibition of action. Germplasm modification Control 00 00ppb -MCP. Temperature & Time Low temperature slows reaction rates by slowing enzyme activity Ethylene perception is slowed and ethylene responses are slowed by low temperature Time is required for product to perceive ethylene and cause response How long an exposure before detrimental effect? Ethylene concentration, temperature, time. Avoidance Keep ethylene sources away from sensitive products Electric forklifts; floor polishers Ethylene producing products Smoking Decaying produce Lettuce Apples

//0. Removal Ventilation about air exchange per hour With fresh air Measure ethylene concentrations to remain below the threshold. Removal Absorption (act. carbon, filters) Oxidation KMnO, U.V., Ozone http://postharvest.ucdavis.edu/yellowpages Ethylene scrubbers and action inhibitors. Inhibition of Production Low temperature Low O Chemical inhibitors of enzymes -AVG, AOA, others Antisense technology -gene/dna inserted reverse order -nonfunctional Natural mutants SAM synthase SAM MET AVG/AOA The Yang Cycle S-methyl thioribose S-methyl thioadenosine Ethylene ACC oxidase - ACC Low O N-malonyl transferase ACC synthase MACC malonyl ACC Molecular manipulation of ripening Anti-sense ACC synthase; Anti-sense ACC oxidase Result - fruits that ripen very slowly, require ethylene treatment to ripen or to produce aroma volatiles Ripening mutants retard the ripening process Example: DFD (Delayed Fruit Deterioration) naturally occurring mutant Respiration, µl CO/g-h 0 0 RESPIRATION 90 X NC (Cantwell, 009) Mutant Wild Type Hybrid =mature-green, =table-ripe COLOR Mutant Wild Type Hybrid Ethylene production, nl/g-h ETHYLENE 0 Mutant Wild Type Hybrid 7 9 0 Days at 0 C Non-modified wild type Anti-sense ACC Oxidase Charentais melons harvested days post-pollination Stored at C for 0 days (J.C. Pech and colleagues) 0 7 9 0 Days at 0 C Cantwell, UC Davis, 009 WT Mutant Hybrid d d

//0 Tomato cultivars and their ripening physiology and ability to ripen Hybrids based on mutants can have greatly retarded ripening off the plant Respiration, µl CO/g-h A. Respiration 0 0 Expt cv Expt cv Expt cv Expt cv Color score C. Red color Expt cv Expt cv Expt cv Expt cv B. Ethylene Ethylene, nl/g-h 0 0 0 Days at 0 C ( F) Tilling technology to select mutants Tilling= Targeting Induced Local Lesions in Genomes 0 0 Days at 0 C ( F) Cantwell, UC Davis, 009 From Kader, UC Davis. Inhibition of action Low O Delays Ripening of Santa Rosa Plums Low temperature high CO low O STS- silver thiosulfate (cut nonedible flowers) Cyclopropenes -MCP (Smartfresh ; Ethylbloc ) Air % O + % CO weeks at 0ºC Months Storage of Bartlett Pears Low O Retards Ripening of Partially Ripe Tomato Fruit Low O -ºC (0ºF) in Air -ºC (0ºF) in % O

//0 CA slows fruit ripening Inhibition of ethylene action -MCP (SmartFresh ) Treatment Gas liberated from a substrate in closed space much like an ethylene treatment Irreversibly binds to ethylene receptor Concentration sufficient to saturate receptors (~000 ppb = ppm) Treatment time sufficient for gas to penetrate tissues (hours) Treatment temperature (0-0 C) Single or multiple applications Product and stage of maturity Ethylene -MCP -MCP - a gaseous ethylene inhibitor Control -MCP Control -MCP Major commercial use to date-keep apples crispy; 0% or more of US apples treated with -MCP 0 C ( F) 0 C (0 F) http://www.agrofresh.com/ cv Bobcat days at 0 C Treated h with -MCP at Breaker stage -MCP Test# to Day delay brown Sequence spots : on hours bananas half-life at retail markets 0 ppb 00 ppb days 0 C Treated at Color stage Control 00 ppb 00 ppb 0 ppb 00 ppb If cannot manage temperature, -MCP could be useful to slow ripening Control fruit. C C 0 C

//0 -MCP & Melons Western shipping cantaloupes-not much benefit on firmness at storage temperature. Eastern shipping cantaloupes-reduce softening at at warm temperatures. Galia; extend shelf-life, reduce firmness loss Watermelon-clear benefit; reduce firmness loss and internal breakdown -MCP, CA and pear ripening From M.S. Reid, UC Davis Fruits and SmartFresh (-MCP) SmartFresh is a powerful regulator of climacteric fruit ripening Its use is complicated for fruit because results depend on: Fruit maturity/ripeness at application -MCP concentration and exposure time Temperature during and following treatment Important to determine where SmartFresh can add value to the fruit category Photos: George Staby EthylBloc sachets (-MCP) in shipping boxes Review Paper: The use of -MCP on fruits and vegetables. C.B. Watkins. 00. Postharvest Biology Technology : 9-09. Russet spot score, =none, 9=severe 7 For many vegetables, -MCP effective for ethylene control but not cost-effective -MCP Prevents Russet Spot Disorder on Iceberg Lettuce Air ppm Ethylene -MCP, Air -MCP, ppm Ethylene LSD.0 Control + Ethylene -MCP + Ethylene. Germplasm selection & engineering Natural variation in susceptibility to ethylene Creation of germplasm insensitive to ethylene 0 0 0 Days at C Test#, midribs; 000 ppb -MCP; Tarraza and Cantwell, 00, UC Davis

//0 Avoiding ethylene effects Selection of resistant varieties Performance of Lettuce Cultivars in Ethylene-induced Russet Spotting ppm ethylene days Russet Spotting Score =none, 9=severe 7 =Sonoma =Spreckles =Salinas =Raider =Salinas 7 =Buena Vista 7=Stinger =Van Sal 0 9=Salinas 0=Ace =Champ =Salinas =Ridgemark A. Iceberg Lettuces B. Romaine Lettuces =Paris Island Cos =Romo =Red Eye Cos =Gx00 =Corazon =Red Hot Cos 7=Nero From M.S. Reid Vanilla Bronze 7 9 0 7 Variety Variety Development of Russet Spot Disorder on Iceberg and Romaine Lettuces. Intact heads were stored days in ppm ethylene at C ( F) plus week in air. The ethylene response cascade Ethylene recognition and signaling pathway. Ethylene response pathway is normally repressed in cells.. When ethylene binds to receptor, inactivates the receptor and allows ethylene response pathway to be active Binding of ethylene inactivates the receptors, resulting in deactivation of CTR, which allows EIN to function as a positive regulator of the ethylene pathway. Etr=ethylene response (receptor) CTR=constitutive-triple-response=a kinase EINS=ethylene insensitive genes ERFs=ethylene responsive factors From M.S. Reid http://mol-biolmasters.masters.grkraj.org/html/gene_expression_ii-plants_more_promoter_elements.htm etr mutation Use of the ethylene receptor gene Arabidopsis Insert mutated gene in sensitive plant becomes insensitive Use tissue specific, or stagespecific promoter ethylene action is inhibited only in that tissue or at that time (e.g. mature flowers, ripe fruits) Already done with petunias, carnations, other flowers Anthony Bleecker9 Transcriptome Changes Associated with Delayed Flower Senescence on Transgenic Petunia by Inducing Expression of etr, a Mutant Ethylene Receptor. H. Wang, G. Stier, J. Lin, G. Liu, Z. Zhang, Y. Chang, M.S. Reid & C Z. Jiang. 0. Plos One (7): e00. 7

//0 New technology use inducible promoters Ethylene has many functions in plants Need to control ethylene in a specific tissue Application of a simple chemical or physical stress Alcohol, dexamethasone, copper, heat Drives synthesis of a gene For example, DEX/etr Ethylene Control Summary Ethylene has both positive and negative effects in postharvest handling of fruits and vegetables Know which fresh products are sensitive to ethylene Do not mix high ethylene producing crops with ethylene sensitive crops Ventilation and low temperatures help control most detrimental ethylene effects Sometimes useful to scrub or oxidize ethylene from storage rooms SmartFresh TM (-MCP) can retard ripening and can be useful for mixed loads Molecular manipulation for specific control From M.S. Reid