Washington Tree Fruit Research Commission

Research Reports

Apple Scald/Lenticel Disorder Chemistry (2002)

FINAL PROJECT REPORT
WTFRC Project #PH-01-120
YEAR 0/0
Organization Project #5798
Title:Apple Scald/Lenticel Disorder Chemistry
PI:John Fellman
Organization:Dept. of Horticulture/Landscape Architecture Washington State University, Pullman, WA, 99164-6414 Tel.: 509-335-3454, e-mail: fellman@wsu.edu
 PDF version of report

Other Personnel

Margo Haines, Postdoctoral Research Associate

Scott Mattinson, Associate in Research,

Graduate Students, Work-Study and Timeslip Employees WSU

Collaborators

James Mattheis, USDA ARS Wenatchee

Professor N.R. Natale, Univ. of Idaho Chemistry Dept

 

Objectives

1) Manufacture and use novel chemical indicators to react with apple skin in order to determine if there is a relationship between chemical indication and scald severity after storage.

2) Determine if the accumulation of chemicals that react to the indicator are related to development of scald symptoms.

3) If (1) and (2) prove feasible, Use the indicators to determine what environmental conditions are implicated in scald development, and investigate non-traditional control methods, the idea being: if we can ameliorate chemical activity, can scald suppression be far behind?

 

Progress to date

Summary of principal findings:
1)      The nitrone spin trapping compound has been successfully manufactured and is now a routine procedure in the Natale laboratory.
2)      Preliminary work showed a trapped free radical related to farnesene as the principal species generated upon UV irradiation of ‘Granny Smith’ apple skin.
3)      UV-Visible spectra show similarity between the reaction generated from UV-treatment, and actual scalded fruit
4)      The nitrone spin trapping compound effectively traps farnesyl free radicals, a result of the farnesene oxidation process leading to scald.
5)      We have identified a trapped free radical related to farnesene as the principal species generated upon UV irradiation of ‘Granny Smith’ apple skin.
6)      UV-Visible spectra show similarity between the reaction generated from UV-treatment, and actual scalded fruit, hence uv-C is an effective “artificial scald“ producer in ‘Granny Smith’.
7)      Absorbance(at 620nm) of the farnesyl-spintrap complex increases as scald symptoms become more severe.
8)      We have tentatively established a “dose-response” relationship between the amount of free radicals generated/detected via spin-trapping and scald severity. 
9)      Our modeling studies reveal that methyl heptenone(MHO), once thought to cause the scald reaction, is merely a signal that the reaction has taken place(confirmed by Ju and Curry(2002) in a recent publication)
10)   Farnesyl Protein Transferase, the hypothetical target of the farnesene hydroperoxide generated(and identified via spin-trapping in the scald reaction) is present in apple tissue.

Narrative—Hypothesis testing and method development

Hypothesis 1: The chemical events resulting from generation of free radicals in apple skin via artificial means are the same chemical events resulting in apple scald.

 

Solvent extraction of reactive components off the fruit surface revealed characteristic spectra of the spin-trapped farnesene radical.  We have concluded that the uv-C treatment of fruit generates relatively smaller amounts of radicalized farnesene. If scalded apples are used, the reactants are apparently still in the apple cuticle at relatively high amounts. We have improved spectral analysis due to purification of the nitrone-farnesyl compound via HPLC. 

 

Our interpretation of the chemical results with regard to the initial radicalization step thought to lead to scald development.  The Farnesyl-nitrone compound is bright red as evidenced by thin-layer chromatography depicted here.  We have obtained a large amount of mass spectrometry information to confirm these observations.

nitrone

Farnesyl adduct

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 1.  (top left) CI-MS of difarnesyl adduct resulting from  reaction of uv-induced farnesene radical and nitrone spintrap; (top right) interpretation of  CI-MS observations; (bottom left) TLC separation of unreacted nitrone from farnesyl adduct.

 

Hypothesis 2: Spin-trapped farnesyl radicals are associated with scald.
Scald Study After 9 Months Storage 2000/2001:Granny Smith apples were harvested at Vantage WA on 9/10/00 and 10/3/00.  The fruit were immediately placed in storage and stored under CA (2.5-3.0% O2, 0.3% CO2) and RA (21% O2, 0.0% CO2).   At this time,the levels of a-farnesene were not detectable in fruit samples.
After 9 months storage in either CA or RA, fruit were removed and sampled for headspace levels of a-farnesene using a dynamic headspace sampling technique with solid-phase-microextraction (PDMS-DVB).  The levels of α-farnesene are shown in figure 3 from fruit after CA harvest 1 and day 0, CA harvest 1 and day 6, CA harvest 2 and day 6, and RA harvest 1 and day 6.  The scald severity was recorded by photographing 25 fruit from each treatment (figures 3-4).  CA fruit after 6 days RT, (figure 3) show obvious scald symptoms.  However, no scald was seen in fruit out of CA and 2-4 hours at RT( figure 4).  The RA fruit after 6 days RT showed the typical bronzing associated with Granny Smith fruit after 21% O2 storage(not shown).The levels of a-farnesene in the CA harvest 1 and day 0 fruit were 2.5 times higher than all other treatments( figure 2).  These fruit had severe scald after 6 days at RT.


Figure 2.  Headspace levels of a-farnesene after 9 months storage in  either CA or RA and the indicated days at room temperature (66oF).

 

Figure 3. Granny Smith H1D6 9 Month Pull CA, 3% O2, 0.3% CO2, 2001

Figure 4.  Granny Smith H1D0, 9 Month Pull, 4 hours from CA, 3.0% O2, 0.3% CO2, 2001

 

 

 

 


Spin-Trap washes and HPLC Purification: In order to detect a-farnesene oxidation products, the nitrone spin trap was washed over the surface of Granny Smith fruit.  Using the nitrone spin-trap at 0.44 g/L in methylene chloride, we applied the spin-trap by washing 1-2 milliliters of solution onto a fruit.  The first treatments required only 3 fruit.  The total volume of wash was 9-10 mls.  The wash(trapped farnesene radicals) was taken to dryness in a rotary evaporator.  The wash was then redissolved  in a uniform volume of methylene chloride. 
Spin-trap bound oxidation products were purified using  high performance liquid chromatography under the following conditions:  5µM spherical silica column (4.0mm X 15cm) ; solvent system of hexane:ethyl acetate at 4:1 in pump A and 100% ethyl acetate in pump B.  The gradient was 0-5 min 100% A then ramp to 30% B over 18 min, after which time the gradient was ramp B to 0% in 7 min; total run time: 25 min.
The results of the first washes from CA and RA treatments (3 fruit) are shown in figure 6.  The RA harvest 1 day 6 fruit after 9 months storage had noticeably higher peaks at 3.5, 4.1, 4.94, and 6.52 minutes (figure 5).  The CA harvest 1 day 6 after 9 months storage had more of the 3.5 minutes peak than other components (figure 5).  The peaks at 3.5 and 6.5 min were collected and characterized by spectrophotometry for each treatment.


Figure 5.  HPLC of nitrone spin-trap rinses from Granny Smith apples.  Each peak indicates free radicals trapped.

A second nitrone spin-trap wash used 25 fruit from each treatment.  The same nitrone spin-trap solution at 0.44 g/L was washed over fruit as previously described.  Approximately 150 ml of wash was collected from each of the treatments, CA harvest 1 day 0, and CA harvest 1 day 6.  Each wash was taken to dryness and redissolved in 2.0 ml of methylene chloride.  Since these 25 fruit washes had a very high level of wax from the fruit surfaces, each sample was slightly heated at 30oC and a 100 µl portion was diluted immediately with 100 µl methylene chloride.  The aliquots were analyzed via HPLC for spin-trapped a-farnesene oxidation products.                                    
Figure 6.  HPLC of nitrone spin-trap rinses from Granny Smith apples.  Each peak indicates free radicals trapped.
The sample from CA harvest 1 day 6 out of 9 months storage had equal levels of all peaks except at 6.52 minutes (figure 6).  The sample from CA harvest 1 day 0 out of 9 months storage had a equal amount of peak 3.5 and 4.1, however, more of peak 4.49 and 4.94 (figure 7).  The peak at 6.52 was lower for the CA harvest 1 day 0 sample (figure 7).  Judging from the 2.5X higher amount of a-farnesene in the CA harvest 1 day 0 sample compared the to CA harvest 1 day 6 sample (figure 2) we would expect more a-farnesene radicals in the washes. 

 

Spectrophotometry of Spin-Trap washes and HPLC Fractions: HPLC fractions from the 3 fruit wash were collected.  Each peak was then analyzed by a HP-8453 diode array spectrophotometer.  The peaks of interest were evaluated for spectrum shifts.  The diode-array spectrum showed an increase at l625nm and l657nm from spin-trap washed fruit peaks.  The absorbance values for RA harvest 1day 6 were 0.447 at l657 compared to CA harvest 1 day 6 at 0.259.  The  unreacted spin-trap had an absorbance value of 0.156 at l657nm.
The HPLC fraction of peak 6.52, from the 25 fruit wash also showed absorbance increases at l 625nm and  l657nm.  The diode-array spectrum is shown in figure 9 from the 25 fruit washes of treatments CA harvest 1 day 6 and CA harvest 1 day 0.  The absorbance value for CA harvest 1 day 0 was 0.323 at l657 nm compared to 0.284 for CA harvest 1 day 6.  This result shows equal levels of spin-trap radicals collected from peak 6.52 in both treatments.  Notice the spin-trap alone of peak 6.52 has a absorbance value of 0.152 at  l657nm.  Thus, upon radical formation and reaction with the spin-trap, an increase at l625 nm as well as l657 nm is observed.
It is concluded that the trapped radicals as evidenced by the bathochromic shift  in the nitrone  are highly correlated to: a) disappearance of  farnesene from the fruit and b) appearance of two trapped species, separated by HPLC.
Hypothesis 3: a) Farnesyl protein transferase exists in apple tissue; b)The presence of farnesene radical(s) affects farnesyl protein transferase.
Farnesyl protein transferase(FPT):  Antibodies to human and tomato FPT, as well as the purified protein from human sources were obtained through generous colleagues in the US and Europe.   Figure 7 shows western-blot analyis of apple tissue.  Two bands at 55 and 66 kDa are seen in tomatoes, but only the 55kDa band is seen apple PEEL preparations when probed with the human antibody. Both bands are seen in petal and stamen preparations indicating developmental differences.  Figure 8 shows western-blot analysis of peel proteins from ‘Granny Smith’ apples, for comparison, a blot stained for total proteins is shown indicating high transfer efficiency

  STD   PET  PET   STA   STA   PL  TOM   FPT   FPT   STD

66kDa

55kDa
97.4

 

66.2
55.0

 

42.7

 


Figure 7. Western blot analysis of apple proteins.  Key: STD: molecular weight standards;PET: petal extract;STA: stamen extract; PL: Granny Smith Peel extract; TOM: tomato extract; FPT: human farnesyl protein transferase.

Figure 8. Western-blot analysis of peel proteins showing the presence of FPT in apples.

 

We conclude that FPT is present in apple peel tissue and may be involved in biological signal transduction.  In the meantime, use of molecular modeling software revealed that farnesyl hydroperoxide, one of the first radicals generated upon farnesene oxidation can interact with the active site of FPT. Figure 9 shows the possible interaction and will be the subject of future work.

 


Figure 9. Model of farnesene hydroperoxide orientation to Tryptophan residue 303 in the active site of FPT.

 


Figure 10. Possible reaction of farnesyl radicals with active site tryptophan-analogy with DPA metabolism in apples


Figure 10 shows the analogy between DPA oxidation and possible FPT active-site tryptophan oxidation.  Our hypothesis 3b needs further investigation.
Hypothesis 4:  The phenomenon noted in ‘Granny Smith’ apples applies to other apples as well.
Scald Study After 9 Months Storage 2001/2002:
Materials and Methods:
1)      ‘Granny Smith’ apples were harvested at Vantage, Washington on 10/5/01.  The fruit were immediately placed into CA storage and stored under CA (1.0% O2, 0.3% CO2) and RA (21% O2, 0.0% CO2) conditions.   Fruit was also stored under different  CA  conditions(5.0% O2, 0.3% CO2) for observation only. 
2)      ‘Gala’ apples were harvested at Vantage, Washington on 8/26/01.  The fruit were stored under RA   (21% O2, 0.0% CO2) or CA (1.0% O2, 0.3% CO2) conditions until evaluated.
3)      ‘Redchief’ Delicious apples were harvested at the Washington State University Tukey Research Orchard on 10/26/01.  The fruit were stored under RA conditions (21% O2, 0.0% CO2). 
Farnesene Analysis:
1)      After 9 months storage in either CA or RA, fruit were removed and sampled for headspace levels of a-farnesene using a dynamic headspace sampling technique with solid-phase-microextration (60 mM PDMS-DVB).  Samples were then injected into a 5890 II Hewlett Packard gas chromatograph with a 5970 mass selective detector.

 

Extraction of Conjugated Trienes from Fruit Wax:
1)      Individual apples were placed in glass beakers of larger diameter, washed for 1min each with 50 mls hexane.  Three fruit from each treatment were pooled according to the method of Rowan et al. (1995).

 

2)      For HPLC analysis an aliquot of 5.0mls hexane rinse was applied to a pre washed 500-mg (3.0 ml) Silica Gel (Bakerbond) SPE extraction column.  The conjugated trienes were washed off the column into a round bottom flask with 2.0 ml of hexane:ether (1:1).  The extract was dried down and redissolved in 2.0 mls of hexane.  At that time the sample was transferred to a 2.0 ml pyrex glass vial with Teflon lined cap (Supelco), and further dried down.  Finally, the sample was reeluted in 1.0 ml HPLC methanol.

 

3)      HPLC was carried out using a Rainin HPLC, UV/VIS system with computer interface.  Analytical conditions were similar to that reported by  Whitaker et al. (1997) using a reverse phase column.  Our system consisted of a C-18 microsorb (Varian Inc.) column, 15 cm X 4.6 with 5mM spheres and 100 Angstrom pores. The sample injected was 20 ul from final extract. The mobile phase was methanol/acetonitrile/water (90:5:5) pumped at 0.8 ml/min.  The conjugated trienols were monitored at l269 nm and farnesene at l232 nm. The conjugated trienol peak that we report is the first peak eluting off the C-18 column and identified according to Rowan, et al. (1995) and Whitaker et al. (1997), as (2,6,10-trimethyldodeca-2, 7(E), 11-tetraen-6-ol). 

 

Spin-Trap washes and HPLC Purification:
1)       In order to detect a-farnesene oxidation products, the nitrone spin trap was washed over the surface of ‘Granny Smith’ fruit.  Using the nitrone spin-trap at 0.44 g/L in methylene chloride, the spintrap was first applied by washing 1-2 milliliters of solution onto a fruit. Our treatments required only 5 fruit.  The total volume of rinse was 9-10 ml.  The rinse was taken to dryness using a rotary evaporator.  The dried rinse was then redissolved in 2.0 ml solvent.  The sample was transferred to a pyrex glass vial with a Teflon lined cap (Supelco). 
2)       To purify the spin-trap bound oxidation products we performed high performance liquid chromatography.  The system was fitted with a silica microsorb (Varian Inc.) column, 15 cm X 4.6 mm with 5 mM spheres and 100 Angstrom pores.  The solvent system was hexane:ethyl acetate at 4:1 in pump A and 100% ethyl acetate in pump B.  The gradient was 0-5 min 100% A then ramp to 30% B over 18 min, after which time the gradient was ramp B to 0% by 25 min.  The amount of sample injected was 10 μl.
Scald Scoring:
1)                  Upon removal from either CA or RA storage, 10 fruit were scored for scald for each treatment.  If individual apple fruits had any visible scald, the score was positive for scald or negative for no scald.  Thus, a portion of 10 fruit was calculated as percentage scald. 
Our results are depicted in figures 11 through 16.  At harvest , a-farnesene levels were slightly detectable in ‘Granny Smith’, yet after 7 months storage, there were copious quantities produced, especially in apples removed from CA conditions(Figure 11).  Conjugated trienol levels were highest in apples taken from RA storage and analyzed immediately(Figure 12). This indicates that farnesene oxidation and generation of organic radicals was taking place.  Two major species of nitrone-trapped radicals related to farnesene oxidation were noted(Figure 13), with the highest levels seen in fruit from RA storage that scalded severely.





 

Figure 11. Farnesene production in  ‘Granny Smith’ after storage.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 12.Conjugated trienol production in ‘Granny Smith’ after storage

 

 

 

 

 

 

 

 

 

Figure 13. Nitrone spintrap farnesyl radicals after CA(top), and RA (middle) storage.  Scald score(bottom) demonstrates relationship between trapped compounds and scald severity.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 14.  Farnesene(top) and conjugated trienol(bottom) production by ‘Redchief Delicious’ and ‘Gala’ after 7 and 9 months RA storage, respectively

 

Figure 14 shows the farnesene-trienol relationship in ‘Redchief’ and ‘Gala’, suggesting a similar phenomenon is a universal occurrence in stored apples regardless of variety.  Interestingly, we measured trapped farnesyl radicals in the skin of both cultivars(Figure 15), yet only ‘Redchief’ displayed scald symptoms(Figure 16), suggesting a natural antioxidant interfering with completion of the scald response in ‘Gala’

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 15. spin-trapped farnesyl radicals and correspondent scald symptoms in ‘Redchief Delicious’ and ‘Gala’

 

 

 

 

 


 

Figure 16. Scald symptoms in ‘Granny Smith’ (left), ‘Redchief Delicious’ and ‘Gala’ (right)

 



It is concluded that the farnesene oxidation phenomenon occurs in all varieties of apples tested, and the differential response to the presence of farnesyl radicals is responsible for differences in scald severity among cultivars. 

General conclusions from this research project.

  1. We can manufacture and use compounds to trap organic free radicals from the surface of fruit skin.  This provides a tool to study oxidative stress events in higher plants.
  2. The presence of two principal farnesyl-nitrone adducts is related to development of scald in apples.  The amount of radicals trapped need to reach a certain level before  symptoms occur.  In some apples (‘Gala’) the radicals appear, yet there are no scald symptoms.
  3. Farnesyl protein transferase is present in apples and modeling studies show that the hydroperoxide resulting from farnesene oxidation may be responsible for inactivation of the transferase protein.

Speculations:
There is a relationship  between oxidation of farnesene, subsequent production of conjugated trienols, and induction of cellular death that gives symptoms of superficial scald.
Chemical evidence suggests Farnesyl Protein Transferase(FPT) is inhibited by active organic peroxides generated by farnesene oxidation
FPT inhibition may be responsible for associated cell death (apoptosis) by preventing posttranslational modification of gene products.

We know:
-          active oxygen species are responsible for  cellular damage
-          farnesene oxidation implicated in scald
-          farnesyl radicals identified in previous work
-          chemical modeling studies show farnesyl hydroperoxide orientation in active site of protein(FPT) distal to tryptophan residue
-          other modeling studies suggest that MHO is NOT a causal agent of scald, a position reinforced by a study recently published by Ju and Curry(2002).
-          DPA is oxidized during interaction with farnesene radicals to prevent scald
-          FPT (as least one form) is present in apple skin
We are trying to ascertain:
-          the exact nature of the two major radicals resulting from farnesene oxidation
-          the characteristics of FPT from apples
-          the relationship between ethylene signaling and scald development

References

Du, Z.Y.  and  W.J. Bramlage 1993.  A modified hypothesis on the role of conjugated trienes in superficial scald development on stored apples. J. Amer. Soc. Hort. Sci. 118:807-813.
Huelin, F.E. and I.M. Coggiola 1970. Superficial scald, a functional disorder of stored apples. VII. Effects of applied α-farnesene, temperature and diphenylamine on scald and the concentration on oxidation of  α-farnesene in the fruit. J. Sci. Food  Agric. 21:584-589.
Ju, Z.  and  E. Curry  2002. Effects of 6-methyl-5-hepten-2-one vapor on peel browning of ‘Delicious’ and ‘Granny Smith’ apples; open vs. closed system. Postharvest Biol. Technol. 25:265-272
Rowan, D. D.; Allen, J. M.; Fielder, S.; Spicer, J. A.; Brimble, M. A. Identification of Conjugated Triene Oxidation Products of  α -Farnesene in Apple     Skin. J. Agric. Food Chem., 1995, 43, 2040-2045.
Whitaker, B. D.; Solomos, T.; Harrison, D. J. Quantification of α-Farnesene and  Its Conjugated Trienol Oxidation Products from Apple Peel By C18-HPLC with  UV Detection. J. Agric. Food Chem., 1997, 45, 760-765.

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