Biochemistry ofCork Formation as a Stress Response

Cândido Pinto RicardoInês Chaves March 5th, 2009

Moore et al. 1998 Botany WCB/McGraw-Hill Companies

Secondary growth of dicot stems

Moore et al. 1998 Botany WCB/McGraw-Hill Companies

Secondary growth of dicot stems Axial section of cork oak tree

Silva et al 2005 International Materials Reviews vol. 6 pp 345

In the cork oak tree, the phellogen forms a continuous layer of cells, enveloping the tree trunk. Each year is produced a 2-3 mm thick layer of cork that adheres to that of the previous year.

A- Cork (Phellem)B- Subero-phellogenic changeC- PhellogeniumD- Liber tissueE- Liber wood changeF- Wood (Xylem)G- BarkH- Lenticular channelI- Area for stopper productionJ- Annual growth rings

www.winomagazine.com/blog2/?cat=8

CorkApplications

http://www.ccrc.uga.edu/~mao/intro/ouline.htm

Structure of plant cell wall

http://www.ccrc.uga.edu/~mao/intro/ouline.htm

T- Tertiary wallS- Secondary wallW- Extractables

(waxes, terpenes, sterols, etc)P- Primary wallM- Middle lamellaPo- Pore

Structure of cork oak cell wall

Silva et al 2005 International Materials Reviews vol. 6 pp 345

Structure of plant cell wall

T SuberinS

W P M

Po

Chemical composition (%):

Suberin 30-50Lignin 15-27Polysaccharides 12-25Extractables (waxes, terpenes, sterols, etc) 8-20Ash 2- 5Others 1- 5

Silva et al 2005 International Materials Reviews vol. 6:345

Cork from cork oak tree

SuberinComplex polymerTwo distinct domains:Polyphenolic and polyaliphatic

Lulai 2007 Skin-set, wound-healing and related effectsin Dick Vreugdenhi Ed. Potato Biology and Biotechnology Advances and Prespectives Elsevier, Amsterdam

Lignin structure

Phenolic precursors

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Shikimate

Triose-P

PEP

Pi

Cytosol

Quinic acid

Quercitol

○

○

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Plastid

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Phenylalanine

Shikimate

Triose-P

PEP

Pi

Chorismate

Cytosol

Quinic acid

Quercitol

○

○

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Arogenate

Tyrosine

Tryptophan

Plastid

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Phenylalanine

Shikimate

p-Coumaric acid

Triose-P

PEP

Pi

Chorismate

Phenylalanine

FlavonoidsCytosol

Quinic acid

Quercitol

○

○

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Arogenate

Tyrosine

Tryptophan

CoumarinsStilbenes

p-Coumaryl alcoolCaffeic acid

Ferulic acid

Sinapic acid

Coniferyl alcool

Sinapyl alcool

Plastid

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Phenylalanine

Shikimate

p-Coumaric acid

Triose-P

PEP

Pi

Chorismate

Phenylalanine

FlavonoidsCytosol

Quinic acid

Quercitol

○

○ LigninsLignans

Cork arom. domain

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Arogenate

Tyrosine

Tryptophan

CoumarinsStilbenes

p-Coumaryl alcoolCaffeic acid

Ferulic acid

Sinapic acid

Coniferyl alcool

Sinapyl alcool

Plastid

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Phenylalanine

Shikimate

p-Coumaric acid

Triose-P

PEP

Pi

Chorismate

Phenylalanine

FlavonoidsCytosol

Quinic acid

Quercitol

○

○ LigninsLignans

Cork arom. domain

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Arogenate

Tyrosine

Tryptophan

CoumarinsStilbenes

p-Coumaryl alcoolCaffeic acid

Ferulic acid

Sinapic acid

Coniferyl alcool

Sinapyl alcool

● DQSDQS

DQS-Dehydroquinate synthase; CM-Chorismate mutase; PAL-Phenylalanine ammonia lyase; CAD-Cinnamoyl alcohol dehydrogenase;CinOR-Cinnamoyl oxiredutase; ChS-Chalcone synthase

● CMCM

●PALPAL

CADCAD●

CinORCinOR●

ChSChS●

Plastid

Aliphatic Precursors of Suberin

KAS- β-Ketoacyl-ACP synthetaseAT - Acyl tranferaseTE - Trans-enoylaseDS - DesaturasePC - Phosphatidyl-cholineTG -Triacilglicerol

Plastid

Endoplasmic reticulum

Plant Lipid Metabolism

1- Two dehydrogenase steps via ω-oxo acid interediate; 2- ω-Hydroxylation; 3- In-chain hydroxylation; 4- Epoxidation of the double bond, followed by hydration5- Fatty acid synthetase; 6- β-Ketoacyl-ACP synthetase II (KAS II) step of fatty acid synthetase; 7- Stearoyl-ACP Δ9-desaturase; 8- Fatty acid elongationACP-Acyl carrier protein

Harwood (1997) Plant Lipid Metabolism in Dey and Harborne Ed. Plant Biochemistry Academic Press, San Diego

Comparative analysis:phellem versus xylem

GenesProteins

Functional categories of genes contributing to cork formation

Soler et al. (2007) A Genomic Approach to Suberin Biosynthesis and Cork Differentiation. Plant Physiology 144:419–431

Differentially Expressed Proteins 3 IEF 10

MM

Carbohydratemetabolism

Energy

Secondarymetabolism Membrane transport

Stress/Defence

Unkown

Regulation/Signalling

10 ºC 28 ºC

15 day

30 day

Stress effects: Air Temperature (10 ºC and 28 ºC)

Triose-PCalvinCycle

Pi

CO2

Erythrose-4-P

Sucrose

PEP

DAHP

Phenylalanine

Shikimate

p-Coumaric acid

Triose-P

PEP

Pi

Chorismate

Phenylalanine

FlavonoidsCytosol

Quinic acid

Quercitol

○

○ LigninsLignans

Cork arom. domain

Glucose-6-P myo- Inositol-1-P

3-DehydroquinateNADH

3-Dehydroshikimate

Arogenate

Tyrosine

Tryptophan

CoumarinsStilbenes

p-Coumaryl alcoolCaffeic acid

Ferulic acid

Sinapic acid

Coniferyl alcool

Sinapyl alcool

● DQSDQS

DQS-Dehydroquinate synthase; CM-Chorismate mutase; PAL-Phenylalanine ammonia lyase; CAD-Cinnamoyl alcohol dehydrogenase;CinOR-Cinnamoyl oxiredutase; ChS-Chalcone synthase

● CMCM

●PALPAL

CADCAD●

CinORCinOR●

ChSChS●

Plastid

The suberized skin of potato tuberas a model to study cork metabolism

Structure of Potato Periderm

Lulai 2007 Skin-set, wound-healing and related effectsin Dick Vreugdenhi Ed. Potato Biology and Biotechnology Advances and Prespectives Elsevier, Amsterdam

Barel, G. et al. J. Exp. Bot. 2008 59:3347-3357

Potato tuber skin development

(A) Number of skin layers (suberized phellem cells) during tuber development

(B) Early stage in periderm development(C) Close-up of dividing phellogen cells(D) Mature skin following foliage removal Bar=200 µm

Cross-sections of tuber surface stained with Safranin O/Fast green and viewed by light (B–D, left panels) and UV (B–D, right panels) microscopy to examine tissue morphology and autofluorescence of suberized cells.

Barel, G. et al. J. Exp. Bot. 2008 59:3347-3357

Representative 2-DE images of skin and tuber storage parenchyma (flesh) at the developmental

stage of 8 weeks post-sprout-emergence

Barel, G. et al. J. Exp. Bot. 2008 59:3347-3357

Cell proliferation Oxidative stressActin (ACT) Ascorbate peroxidase 1 (APX1), cytosolicP23 tumor protein-like (P23/TCTP) Catalase isozyme 2 (CAT2)Proteasome {alpha}-7 subunit Catechol oxidase B, chloroplast precursorProteasome β-2A subunit Polyphenol oxidase (PPO)Translation init iation factor 5A-3Tubulin {alpha}-chain Plant defenceSignal transduction—cell wall Cysteine protease 1 (CYP1)Remorin (REM) Elicitor-inducible protein EIG-J7

Elicitor-inducible protein EIG-J7General metabolism Endochitinase 2 precursorUDP-glucose:protein transglucosylase (UPTG2) Endochitinase 2 precursorDisulphide-isomerase protein (PDI) PatatinTriosephosphate isomerase, (TPI) cytosolic isoform Patatin putative homologOxidative respiratory chain Patatin protein 07APFI (hypothetical protein F8G22.2) Pathogenesis-related protein 10 (PR-10)NADH-ubiquinone oxidoreductase 18 kDa subunit Pathogenesis-related protein 10 (PR-10)NADH:FMN oxidoreductase-like protein 2-Oxoglutarate-dependent dioxygenase (SPP2)

One-carbon (C1) metabolism Suberization/lignificationGlutamate-ammonia ligase (GS1) ACP-17 kDa β-hydroxyacyl-acyl carrier proteinSerine hydroxymethyltransferase 4 (SHMT4) Caffeoyl-CoA O-methyltransferase-5 (CCoAOMT-5)Methionine synthase (MS) Caffeoyl-CoA O-methyltransferase-6 (CCoAOMT-6)Abiotic and biotic stress Caffeoyl-CoA O-methyltransferase-3 (CCoAOMT-3)Plasma-membrane polypeptide (DREPP) Peroxidase (POD 18)

Peroxidase PER9-6 secretory (POD 20)Reference protein Peroxidase 136, class III , precursor (POD 9)Nascent polypeptide-associated complex NAC; UBA-like Peroxidase putative (POD 5)

Peroxidase, suberization-associated anionic peroxidase

List of proteins that accumulate differentially in potato tuber skin compared to tuber storage parenchyma

EVALUATION OF WOUND-HEALING PROCESSES IN POTATO TUBER TISSUE

Potato Slices

Day 0 Day 8Day 4

Potato Slices

Day 0 Day 8Day 4

Cellular dediferentiationGene expressionOxidative stress response

Suberin deposition

Wounding - Wound response - Healing

Periderm formation

Potato Slices

Day 0 Day 8Day 4

Cellular dediferentiationGene expressionOxidative stress response

Suberin deposition

Suberin detected

Wounding - Wound response - Healing

Periderm formation

Time-course of peroxidase and oxidase activity in wound-healing potato tubers

NADPH-dependent O2.– generation

Peroxidase activity

Razem et al. J.Exp. Botany, Vol. 54:935-941

Proteomics of the wound-healing process

3 10pI

Clustered mean expression profiles of differentially expressed proteins

Clustered mean expression profiles of differentially expressed proteins

Wound response

Clustered mean expression profiles of differentially expressed proteins

Periderm reconstruction

Clustered mean expression profiles of differentially expressed proteins

Periderm reinforcement

Cork FormationCellular Processes• Phellogen proliferation • Phellogen derived cells

– Phellem commitment– Cell expansion– Cell senescence– Suberin biosynthesis and waxes deposition– Cell death

Biochemical Processes• Cork results from 4 main secondary metabolic pathways:

– Acyl-lipids (aliphatic suberin domain)– Phenylpropanoids (cork aromatic components)– Isoprenoids (wax terpenes and sterols)– Flavonoids (tannins)

• Peroxidase activity fundamental (presence of H2O2)• Integration of biochemical process almost unkown