WP5 Development of a strategy for the selection of varieties with good storage behaviour DLO Renata Ariens, Jurriaan Mes, Jan Verschoor

Objective

• New varieties should have a good storage potential

• Practice: almost year round storage at Controlled Atmosphere conditions (ULO, DCA)

• Problems: chilling injury, CO2 damage, decay, loss of firmness, short shelf-life when taken from storage

• Develop a test which can select good varieties early during breeding and selection: Storage Stress Test

Stage 1. Selection of storage conditions

Five commercially grown cultivars • Cox O.P. • Elstar • Golden Delicious • Jonagold • Kanzi Harvested (2012) • a week before • at commercial harvest date (for long term ULO-storage) • 1 week after Storage period • 1 week conditioning + 1 week shelf life • 1 month conditioning + 1 wk. shelf life • 2 months conditioning + 1 wk. shelf life

regime T (°C) %O2 %CO2

1 -1 21 0

2 -1 1 5

3 10 21 0

4 10 1 5

Number of apples per time point scored 20

Parameters analysed

• At harvest before storage

– Starch

– Firmness (penetrometer, aweta)

– Colour

– DA meter

– Titratable Acids

– Brix

• After storage and shelf-life

– Firmness

– Colour

– Chilling injury (index)

– CO2 damage (index)

Cultivar behaviour in test

Cultivar

Max storage period

(optimal conditio

ns)

Low Temp Sensitivity flesh

browning Texture issues

Low O2/high CO2 tolerance

Disorder sensitivity

practice

test practice test practice test practice test

Cox 5-6 m +++

+++

(-1°C + CA, late harvest, ≥1 month)

Can develop mealiness

before softening

Mealiness , rapid

softening (air,

≥1 month)

- - Bitter pit,

brown core, Softening

Kanzi 12 m +++

++++

(-1°C + CA, late harvest, 2

months)

Very firm, tough peel

Very firm indeed

++

++

(-1°C + CA, last harvest, ≥1 month)

bitter pit, lenticel

breakdown

Jonagold 9 m - - Greasiness Greasiness (10°C, air, ≥1

month)

++

+ (-1°C + CA,

3 harvest, ≥1 month)

Scald, flesh browning

Golden 8 m - -

- Greasiness (10°C, air, ≥1

month) ++

++

(-1°C + CA, 1+3 harvest, ≥1 month)

Scald

Elstar 7 m + - Rapid

softening after storage

- + - Softening, skin spots

Skin spots (all treatm

Conclusion from Y1

• Chilling injury is reflected at -1˚C +CA, late harvest, >1 month storage

• CO2 sensitivity is reflected at -1˚C +CA, late harvest, >1 month storage

• Texture issues are best reflected at (10˚C) air, all harvest, >1 month storage

• Disorders, difficult to induce, symptoms very rare

• Minimum fruits needed ~ 40, for 1 stress test

Y 2

Objective: expand the correlation analysis between cultivar storage behaviour and stress test

– 13 cultivars (INRA)

– Harvest 2013

– Stored for 1 month at -1°C in CA (1%O2/5%CO2)

Analysis at start

- Firmness (FTA automated penetrometer, destructive)

- Ground colour (colour chart)

Analysis after storage and shelf-life

- Firmness (FTA automated penetrometer, destructive)

- Ground colour (colour chart)

- External and internal disorders

(expert visual evaluation)

Experimental setup

• 13 cultivars from INRA Angers

• harvest date determined by INRA (starch index 7)

• Randomized after harvest

planning for each cultivar day

harvest (INRA) 0 holding temperature day 0-7: 4°C

transport to FBR 1-6 courier transport between day 1-6 (<10h)

storage at FBR 7 storage duration 35 days

storage at 2 regimes:

4°C in air (= control)

-1°C in CA [1%O2/5%CO2] (= stress inducing

treatment)

shelf life 42 7 days at 18°C/80% RH

quality check & freezing

RNA-sample 49

Stress test material

cultivar harvest days between harvest and transport to

FBR

Cox 20-sep 12

Elstar 26-sep 6

Greensleave 26-sep 6

Canada rouge 26-sep 6

Gala face A 26-sep 6

Gala face B 26-sep 6

Golden 26-sep 6

Ariane 26-sep 6

Jonagold 26-sep 6

Belle de Boskoop 26-sep 6

Chanteclerc 18-okt 18

Fuji 23-okt 13

Granny Smith 31-okt 5

Was not possible to have it standardized among samples

Stress test results: Firmness

Average of firmness (kg) storage regime

cultivar -1°C in CA (1%O2/5%CO2) 4°C in air Grand Total

Chantecler 4.63 3.10 3.87

Greensleeve 4.20 3.57 3.88

Canada Rouge 6.47 4.36 5.41

Cox 6.32 4.58 5.42

Jonagold 6.75 4.57 5.63

Elstar 6.19 5.26 5.72

Golden 7.35 4.10 5.72

Gala Face B 7.07 5.61 6.34

Gala Face A 7.05 5.69 6.37

Belle de Boskoop 8.43 5.69 7.06

Fuji 7.55 7.29 7.42

Granny 7.86 7.83 7.84

Ariane 9.23 8.52 8.87

Grand Total 6.87 5.42 6.14

Stress test results: Colour

Average of colour-index storage regime

cultivar -1°C in CA (1%O2/5%CO2) 4°C in air Grand Total

Chantecler 4.9 5.0 4.9

Cox 4.6 4.8 4.7

Gala Face A 4.0 4.8 4.4

Jonagold 3.9 4.3 4.1

Canada Rouge 4.0 4.1 4.0

Gala Face B 3.9 4.1 4.0

Greensleeve 3.7 4.3 4.0

Elstar 3.6 3.9 3.8

Golden 3.1 4.1 3.6

Fuji 3.6 3.6 3.6

Ariane 3.0 3.8 3.4

Belle de Boskoop 3.0 3.1 3.1

Granny 2.0 2.4 2.2

Grand Total 3.6 4.0 3.8

Hardly disorders inducted

Only in -1°C in CA (1%O2/5%CO2)+ shelflife

cultivar Internal CO2-injury (% of fruits) Chilling injury (% of fruits)

Ariane

Belle de Boskoop

Canada Rouge 25% 70%

Chantecler

Cox

Elstar

Fuji 10%

Gala Face A

Gala Face B

Golden

Granny

Greensleeve

Jonagold

Conclusion Y2

• Difficult control harvest time

• Limited disorder symptoms

• Due to pre-storage (4˚C) conditions ?

• 1 months storage not enough ?

• Year to year variation ?

Can gene expression marker speed up storage behaviour diagnose

• Stress test better performed for up to 3 months

– More expensive

– Results later in time and next experiments can be planned

• Do changes in gene expression already predict those cultivars that have storage problems

Experiment 3

- Harvest 2014

- 4 cultivars x 5 different growers (NL)

- Late harvest

- stored at -1°C in CA (1%O2/5%CO2)

- Storage duration 3.5 months (+ 1 wk shelf life)

- Take apple sample after 1 wk for gene expression analysis

- Relate these genes with storage after 3.5 months

0.00

2.00

4.00

6.00

8.00

10.00

12.00

pe

ne

tro

me

terv

alu

e (

kg)

Firmness

at harvest after 4 months stess inducing treatment (-1°C/1%O2/5%CO2) + 1 week 18°C

0%

5%

10%

15%

20%

25%

30%

35%

40%

External CO2 injury-index (0-100%)

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

internal CO2-injury

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Average of Chilling injury

Behaviour in practice and test

Cultivar

Max storage period

(optimal conditions)

Low Temp Sensitivity flesh

browning Texture issues

Low O2/high CO2 tolerance

Disorder sensitivity

practice test practice test practice test practice test

Kanzi 12 m +++ ++

Very firm, tough peel

Very firm indeed

+ ++/+

bitter pit, lenticel

breakdown -

Belle de Boskoop

6 m +++ +++ Softening Softening but

acceptable + -/++

Scald, flesh browning

-

Golden 8 m - -

- Softening but acceptable

++ - Scald -

Elstar 7 m +/- +

Rapid softening

after storage

Softening but

acceptable -

+/- +/+ Softening, skin spots

Skin spots (all treatm

Conclusions all tests

• Chilling injury good inducible

• CO2 injury more difficult to induce in agreement with practice (depending of specific pre harvest conditions?)

• Except for skin spot, disorders hard to induce by this storage stress test

• Can we then study varieties better supported by gene expression level as predictive biomarkers?

Samples for microarray

Cultivar

Genetic background

Max storage period

(optimal conditions)

Texture issues Low Temp

Sensitivity flesh browning

Low O2/high CO2 tolerance

Disorder sensitivity

practice test practice test practice test

Cox

5-6 m Can develop

mealiness before softening

+ +++ ++ - - Bitter pit, brown core, Softening

Golden 8 m +/- - - ++ + Scald

12 arrays INRA, dye swap 6 samples selected 1 wk storage samples, to predict effect of longer storage

Sample for array

start 1 wk -1˚C air 1 wk -1˚C CA

Cox Cox A CoxB Chilling stress

CoxD Chilling stress, less CO2 stress

Golden D GDA GDB No chilling stress

GBD No chilling stress, only CO2 stress

Cox D (cold and CO2) Cox B (cold)

Red= upregulated Blue= down regulated

Redox down regulated Peroxidase up Signalling up Cell wall up Beta glucanase up

Redox is most up Peroxidase down Signalling down regulated Cell wall down regulated

Cox D (cold and CO2): Transport:

aa transport Redox:

ascorbate + glutathione Cell wall:

degradation and modification Cox B (cold) Misc:

cytochrome p450, gluco-galacto-mannosidases, β 1,3 glucan hydrolases, peroxidases

Lipid metabolism:

FA synthesis + elongation and lipid transfer proteins

Data set used is t-test between air and CA storage

Bin name description Cox A Cox B Cox D

cell wall.degradation.mannan-xylose-arabinose-fucoseat4g30270Symbols: MERI5B, MERI-5, SEN4 | MERI5B (meristem-5); hydrolase, acting on glycosyl bonds / xyloglucan:xyloglucosyl transferase | chr4:14819192-14820592 REVERSE0.972 -0.438 -3.058

misc.peroxidases at5g05340peroxidase, putative | chr5:1578951-1580875 REVERSE 1.281 0.768 -2.440

signalling.calcium at3g22910calcium-transporting ATPase, plasma membrane-type, putative / Ca(2+)-ATPase, putative (ACA13) | chr3:8116335-8119388 REVERSE0.365 0.777 -1.175

Bin name description Cox A Cox B Cox D

RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element binding protein familyat1g22810AP2 domain-containing transcription factor, putative | chr1:8074231-8075037 REVERSE0.277 -0.148 0.519

signalling.receptor kinases.wall associated kinaseat1g21230Symbols: WAK5 | WAK5 (WALL ASSOCIATED KINASE 5); kinase/ protein serine/threonine kinase | chr1:7429980-7432346 FORWARD-0.121 0.475 0.731

fermentation.ADH at1g77120Symbols: ADH1, ADH, ATADH, ATADH1 | ADH1 (ALCOHOL DEHYDROGENASE 1); alcohol dehydrogenase | chr1:28975394-28977417 FORWARD-0.676 -1.266 1.123

hormone metabolism.ethylene.signal transductionat4g31980unknown protein | chr4:15464905-15469204 FORWARD 0.613914 -0.00336 0.53954

cell wall.cell wall proteins.RGP at5g16510reversibly glycosylated polypeptide, putative | chr5:5392793-5394568 FORWARD-0.32762 0.293879 0.558189

misc.short chain dehydrogenase/reductase (SDR)at3g51680short-chain dehydrogenase/reductase (SDR) family protein | chr3:19173622-19174667 REVERSE0.709022 0.208936 0.651472

Bin name description Cox A Cox B Cox D

cell wall.cellulose synthesis at4g07960Symbols: ATCSLC12, CSLC12 | ATCSLC12 (CELLULOSE-SYNTHASE LIKE C12); cellulose synthase/ transferase, transferring glycosyl groups | chr4:4802341-4805647 REVERSE-2.332 -0.534 -1.238

RNA.regulation of transcription.WRKY domain transcription factor familyat1g62300Symbols: WRKY6, ATWRKY6 | WRKY6; transcription factor | chr1:23016683-23019279 REVERSE-2.420 -0.323 -1.049

lipid metabolism.FA synthesis and FA elongation.ACP desaturaseat1g43800acyl-(acyl-carrier-protein) desaturase, putative / stearoyl-ACP desaturase, putative | chr1:16577610-16579722 FORWARD-2.749 -2.224 1.437

Possible stress markers for Cox

10 differential genes found in analysis and also present in literature

Differential expression GD

PCA Golden delicious 1% van genen

GDB= -1°C air

GDD= -1° CA

GD B ( cold) GD D (cold and CO2)

Beta-glucanase= cryo/cold protective

Peroxidases react to high CO2= scavenging radicals

Red= upregulated Blue= down regulated

GD B (cold) Transport:

oa unspecified cations, aa, NDP sugars at the ER

Protein: synthesis, posttranslation modification, degradation

RNA: regulation of transcripts

Stress: biotic PR protein

AA metabolism: synthesis, aromatic aa

Lipid metabolism: exotics oa steroids, squalene and lipid degradation

GD D (cold and CO2) Redox:

ascorbate +glutathion and thioredoxin

C1 metabolism: glycine hydroxymethyltransferase

Dataset used is t-test between air and CA storage

Bin name description GD A GD B GD D

lipid metabolism.''exotics'' (steroids, squalene etc).sphingolipidsat3g61580delta-8 sphingolipid desaturase (SLD1) | chr3:22786047-22787972 FORWARD1.832 -5.553 -2.251

misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidaseat1g64760glycosyl hydrolase family 17 protein | chr1:24054173-24056414 REVERSE0.530 -4.854 -1.390

cell wall.degradation.cellulases and beta -1,4-glucanasesat1g19940Symbols: AtGH9B5 | AtGH9B5 (Arabidopsis thaliana Glycosyl Hydrolase 9B5); catalytic/ hydrolase, hydrolyzing O-glycosyl compounds | chr1:6918191-6920377 REVERSE1.171 -4.761 -1.890

cell wall.degradation.pectate lyases and polygalacturonasesat4g23820glycoside hydrolase family 28 protein / polygalacturonase (pectinase) family protein | chr4:12397038-12400091 REVERSE0.373 -4.344 -1.692

misc.peroxidases at5g05340peroxidase, putative | chr5:1578951-1580875 REVERSE -1.155 -3.333 -2.265

Bin name description GD A GD B GD D

cell wall.cell wall proteins at3g48530Symbols: KING1 | KING1 (SNF1-RELATED PROTEIN KINASE REGULATORY SUBUNIT GAMMA 1) | chr3:17987378-17989763 FORWARD-0.214 -3.963 0.436

amino acid metabolism.degradation.aromatic aa.tryptophanat5g65940Symbols: CHY1 | CHY1 (BETA-HYDROXYISOBUTYRYL-COA HYDROLASE 1); 3-hydroxyisobutyryl-CoA hydrolase | chr5:26376610-26379235 REVERSE0.145 -3.819 0.422

lipid metabolism.''exotics'' (steroids, squalene etc).sphingolipidsat1g27980Symbols: DPL1, ATDPL1 | DPL1; carboxy-lyase/ catalytic/ pyridoxal phosphate binding | chr1:9748737-9752769 FORWARD0.203 -3.689 0.542

hormone metabolism.gibberelin.synthesis-degradation.ent-kaurenoic acid hydroxylase/oxygenaseat2g32440Symbols: KAO2, CYP88A4 | KAO2 (ENT-KAURENOIC ACID HYDROXYLASE 2); ent-kaurenoate oxidase/ oxygen binding | chr2:13775570-13778002 FORWARD0.511 -3.646 1.499

RNA.regulation of transcription.WRKY domain transcription factor familyat4g31550Symbols: WRKY11, ATWRKY11 | WRKY11; calmodulin binding / transcription factor | chr4:15289947-15291539 REVERSE0.360 -3.616 0.505

cell wall.cellulose synthesis.cellulose synthaseat4g18780Symbols: CESA8, IRX1, ATCESA8, LEW2 | IRX1 (IRREGULAR XYLEM 1); cellulose synthase/ transferase, transferring glycosyl groups | chr4:10312624-10316790 REVERSE1.000 -3.431 0.513

signalling.calcium at4g38810calcium-binding EF hand family protein | chr4:18115283-18119077 REVERSE0.469 -3.369 0.491

cell wall.pectin*esterases.PME at3g29090Symbols: PME31, ATPME31 | pectinesterase family protein | chr3:11073703-11075478 FORWARD0.932 -2.785 0.372

Bin name description GD A GD B GD D

misc.beta 1,3 glucan hydrolases at5g55180glycosyl hydrolase family 17 protein | chr5:22388782-22390714 FORWARD-0.410 1.554 0.445

RNA.regulation of transcription.bZIP transcription factor familyat5g60830Symbols: AtbZIP70 | AtbZIP70 (Arabidopsis thaliana basic leucine-zipper 70); DNA binding / transcription factor | chr5:24472639-24473319 FORWARD-1.615 1.593 -0.530

misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidaseat5g20340Symbols: BG5 | BG5 (beta-1,3-glucanase 5); glucan 1,3-beta-glucosidase/ hydrolase, hydrolyzing O-glycosyl compounds | chr5:6874787-6875851 FORWARD-1.473 1.712 -0.053

stress.biotic at4g33720pathogenesis-related protein, putative | chr4:16182752-16183533 FORWARD0.154 2.300 0.219

Possible stress markers for GD

12 differential genes from the different tests and also in literature

Ethylene signalling pathway

P.A Wiersma et al Post Harvest Biology and Technology 44 (2007

Methionine + ATP

S-adenosyl methionine (SAM)

ACC-synthase (9)

Md-ACS1, Md-ACS2, Md-ACS3, Md-ACS5

1-aminocyclopropane-1-carboxylic acid (ACC)

ACC-oxidase (5)

Md-ACO1, Md-ACO2, Md-ACO3, md-ACO4

Ethylene [C2H4]

Receptors (5) Md-ETR1 Md-ERS1 Md-ETR2 Md-ETR5 Md-ETR1b Md-ERS2

AF032448 AY083169 DQ847145 DQ847146 AY821544 AB213028

CTR1 (1) Md-CTR1

AY670703

EIN2 (1) Md-EIN2a Md-EIN2b

CV084545 CO066480

EIN3/EIL (6) Md-EIL1 Md-EIL2 Md-EIL3 uit E. Tacken Plant Physiol. 153 (2010)

GU732484 GU732485 GU732486

ERF Md-ERF1 Md-ERF2 Md-ERF3 Md-ERF4 Md-ERF5 Md-ERF6

AB288347 AB288348 JF412350 JF412351 JF412352 JF412353

uit A. Wang J.Exp.Botany (2007) uit A. Akagi Post Harv.Path.and Mycotoxins (2011)

expressed in fruit

What happens with Ethylene pathway in microarray exp

Ethylene signalling pathway Genes ethylene signalling are checked for allelic variation

Golden delicious genome

RNA seq data Cox and Breaburn

alignment of RNA seq data to golden delicious genome

check for SNP and other variation

• Design qPCR primers on specific conserved regions for each gene

• Some genes have variation compared to GD, consequence of these variations is still being analysed

Can we predict ?

• Perform qPCR on material after 1 wk stress test + 1 wk shelf-life

• Correlate to quality parameters of samples after 3.5 months stress test and 1 wk shelf-life

• Ongoing

Conclusions

• 2-3 months storage at -1°C in CA (1%O2/5%CO2) + 1 wk shelf life can be selective

• GD and Cox behave differently in first week of storage stress

• Several genes selected from known pathway and functions, incl ethylene related genes (EIL1, ERF2)

• Now study correlation among 3 samples of exp 1 and 3 to relate storage potential with gene expression

Wrap-up

– Stress test has potential for screening postharvest behaviour prediction

– Needs further optimisation / standardisation

– Marker selection shows potential

– Last analyses ongoing

Thanks for your attention