1. Understanding Postharvest Abscission in Balsam Fir Dr. Mason T. MacDonald Sept 8, 2012

3. Introduction Who am I? ▫Started PhD project working with balsam fir in 2007 ▫Graduated 2010 ▫Started postdoctoral research at CRC in 2010 ▫Currently managing the uplc-ms research lab ▫Over a dozen publications regarding senescence/abscission

4. Introduction What do I work on? ▫“Officially” – the artificial root technology (ART) platform ▫Unofficially – everything Today I’ll be discussing known signals for abscission in balsam fir and how it’s linked to conventional understanding.

5. Problem Post-harvest needle abscission ▫Healthy branch/tree cut down ▫Something happens to it (?) ▫Needle abscission occurs Objective: find out what happens after it is cut

6. Ethylene-induced abscission Typical role of ethylene in other species: ▫Endogenous ethylene peaks before abscission ▫Exogenous ethylene will trigger abscission ▫Inhibition of ethylene action will prevent abscission Picture of carnations on the left, snap dragon on the right. Both pictures were taken by Michael Reid at University of California, Davis, CA.

7. Objectives To understand the role of ethylene in needle abscission of root-detached balsam fir More specifically: 1.To uncover the role of exo- and endogenous ethylene 2.To confirm the role of ethylene using ethylene inhibitors 3.To describe the action of ethylene on cellulase activity 4.To uncover the biophysical trigger for ethylene

8. Sample Collection Tree breeding center ▫Located in Debert, NS ▫Access to grafted clones ▫Branch with 2 year growth served as experimental unit ▫Samples were cut from the SE side of a tree 1.5 m above ground ▫Samples placed in water and transported to growth chamber Growth chamber conditions ▫22 °C at day, 15 °C at night ▫50% humidity ▫80 µmolm -2 s -1

9. Branch Protocol 1. Branches were given a fresh cut and wrapped in cotton wool 2. Branches were placed in a flask and provided 200 mL water. Entire unit was weighed 3. Each unit was placed in an ethylene incubation chamber

10. Experiment 1: DurationConc. (ppm) 24 hours0 10 100 500 1000 Continuous0 10 100 500 1000 Exogenous ethylene ▫40 branches sampled ▫4 replicates in each treatment ▫NRD, AWU, and XPP measured Endogenous ethylene ▫70 branches sampled ▫No treatments ▫NRD, DPE, and ethylene evolution measured Experimental design to test the effect of exogenous ethylene on balsam fir abscission Experiment 1 To uncover the role of exo- and endogenous ethylene

11. Short-term exposure results After a 24-hour ethylene exposure duration: ▫50 to 100% increase in NRD ▫No change in AWU or XPP Experiment 1

12. Continuous exposure results CONTROL Experiment 1

13. Continuous exposure results 1000 ppm ETHYLENE Experiment 1

14. Continuous exposure results After continuous exposure to ethylene: ▫Up to 60% decrease in NRD ▫Up to 80% increase in AWU ▫Up to 160% decrease in XPP Day 0Day 8Day 11Day 13 Experiment 1

15. Endogenous ethylene evolution Ethylene detectable at day 12 ▫2 days before needle abscission starts Ethylene peaks at day 22 ▫2 days before needle abscission is completed Experiment 1

16. Conclusions Physiological: ▫Endogenous ethylene increases after harvest and peaks immediately prior to abscission ▫Continuous exposure to concentrations as low as 10 ppm will induce abscission Practical ▫24-hour exposure to relatively low concentrations of ethylene will delay abscission Experiment 1

17. AVG 1-MCP

18. Experiment 2: AVG (inhibits ethylene synthesis) ▫48 branches sampled ▫2 x 6 factorial ▫4 replicates Factor 1: Ethylene conc. ▫0 or 1000 ppm Factor 2: AVG conc. ▫0, 1, 10, 100, 500, or 1000 ppm 1-MCP (blocks ethylene receptors) ▫40 branches samples ▫2 x 5 factorial ▫4 replicates Factor 1: Ethylene conc. ▫0 or 1000 ppm Factor 2: 1-MCP mass used ▫0, 2.5, 5.0, 7.5, or 10.0 g Experiment 2 To confirm the role of ethylene using ethylene inhibitors

19. Inhibiting ethylene action Experiment 2

20. Inhibiting ethylene action Day 0Day 40 Control1000 ppm AVG 10 g 1-MCP Control1000 ppm AVG 10 g 1-MCP

21. Conclusions Physiological: ▫Inhibiting ethylene action increases NRD ▫Further supports that ethylene is signal required for abscission Practical: ▫Both products could easily be used by producers ▫AVG could be used by the consumer (i.e. dissolve in water supplied to tree) Experiment 2

22. Experiment 3: Control (no signs of abscission) ▫0 ppm exogenous ethylene ▫Protein extracted at day 14 Endogenous ethylene-induced abscission ▫0 ppm exogenous ethylene ▫Protein extracted at day 35 Exogenous ethylene-induced abscission ▫1000 ppm exogenous ethylene ▫Protein extracted at day 14 Experiment 3 To describe the action of ethylene on cellulase activity

23. Results Molecular weight LowerHigher 250 kDa 20 kDa Cellulase Standard Exogenous ethylene Endogenous ethylene No abscission control C2H4C2H4 Control Experiment 3

24. Conclusions Physiological ▫Cellulase increases nearly 10-fold prior to abscission with either endogenous or exogenous ethylene Experiment 3

25. Experiment 4: Throughout the experiments, common results have been noticed with respect to AWU and XPP Experiment 4 To uncover the biophysical trigger for ethylene

26. Design Modifying storage humidity ▫2 x 3 factorial ▫Ethylene (0 or 1000 ppm) ▫Humidity: (30, 60, or 90 %) ▫4 replicates Modifying storage temperature ▫2 x 3 factorial ▫Ethylene (0 or 1000 ppm) ▫Temperature: (5, 15, or 25 °C) ▫4 replicates Experiment 5 Response variables were NRD, AWU, XPP, DPE, and ethylene evolution in both experiments

27. Temperature Results 5 °C was the most effective ▫40% decrease in AWU ▫XPP half of higher temperatures ▫NRD doubled Experiment 5 Ethylene (ppm) Temperature (°C) NRD (days) AWU (mLg -1 d -1 ) XPP (MPa) DPE (days) C 2 H 2 Evolution (µmolg -1 h -1 ) 05 65.0 a 0.035 c -0.34 c 61.5 a 4.9 b 15 37.5 b 0.045 b -0.64 b 34.5 b 4.7 b 25 29.5 c 0.058 a -0.76 ab 27.8 c 7.4 a 10005 20.8 d 0.032 c -0.51 b 18.0 d 5.3 b 15 13.3 e 0.042 bc -0.60 b 11.5 e 5.8 b 25 11.0 e 0.065 a -0.90 a 9.5 e 8.0 a

28. Humidity Results 90% humidity was the most effective ▫Almost 70% decrease in AWU ▫XPP only -0.09 MPa in absence of ethylene ▫NRD increased by more than 5 times! Experiment 5 Ethylene (ppm) Humidity (%) NRD (days) AWU (mLg -1 d -1 ) XPP (MPa) DPE (days) C 2 H 2 Evolution (µmolg -1 h -1 ) 030 28.5 c 0.061 a -0.73 b 27.0 c 7.5 60 32.3 b 0.038 b -0.40 c 30.5 b 6.4 90 149.8 a 0.021 c -0.09 d 145.8 a 6.1 100030 11.8 d 0.058 a -0.94 a 10.3 d 5.8 60 12.0 d 0.041 b -0.84 a 10.3 d 5.4 90 13.8 d 0.029 bc -0.81 a 12.3 d 7.1

29. Humidity Results Experiment 5 Day 1Day 30Day 60 30%90%30%90%30%90% Day 90

30. Humidity Results VPD: the difference between amount of moisture in the air and what it can hold

31. Conclusions Physiological ▫Minimizing water use delays ethylene evolution and increases NRD ▫Decreasing water potential may be the trigger to begin abscission Practical ▫Transport and/or storage at low temperature and high humidity could improve product Experiment 5

32. Discussion Ethylene is strongly implicated as abscission signal molecule in balsam fir Five methods to delay needle abscission ▫24-hr ethylene exposure ▫Ethylene synthesis inhibition (AVG) ▫Ethylene receptor blockage (1-MCP) ▫Low temperature storage ▫High humidity storage

33. Hormonal Signals Ethylene is just one hormone. What is happening with other hormones? Objective: compare hormone concentrations before and during abscission Samples taken from trees in the field ▫Immediately frozen in liquid N to halt physiological activity Branches cut, placed in water ▫Needles collected during abscission, frozen and prepared for analysis

34. Hormone Profile - ABA ABA and derivatives all increased ABA increased 38 times PA increased 52 times DPA increased 90 times

35. Hormone Profile - Cytokinins Most cytokinins doubled 4-fold increase in t-ZR

36. Hormone Profile - Auxins 95% decrease in IAA related to ethylene abscission response

37. Temporal Changes - Results Needle abscission increased rapidly after 20 days Changes in abscission correspond with changes in capacitance

38. Temporal Changes - Results Several response variables changed immediately before rapid abscission Similar results in branches with and without water (only difference was timing) ParameterInitialFinal XPP (MPa)- 0.2- 0.7 Capacitance (nF)7.02.0 RWC (%)8050 Break Strength (N)1.50.5 MII (%)412

39. What Do We Know? Root DetachmentTrigger (s) ? Increased cellulaseAbscission Drying (↓XPP, RWC, capacitance) Hormonal (↓Auxin, ↑cytokinins, ABA) Ethylene Evolution Ethylene Receptors AVG 1-MCP Adjust VPD

40. My daughter seeing her first Christmas tree