Presentación de PowerPoint · molecular markers (physiological, genetic or epigenetic hallmarks) of ageing and reinvigoration understanding of the molecular basis of ageing and reinvigoration

AGEING, A BARRIER IN FORESTRY PRODUCTION

↓ ↓ ↓ CLONAL MULTIPLICATION CAPACITY

COMPLEX ARCHITECTURE↓ ROOTING↓ MORPHOGENIC ABILITY↓ IN VITRO ESTABLISHMENT

REPRODUCTIVE GROWTH

↑↑ ↑ CLONAL MULTIPLICATION CAPACITY

SIMPLE ARCHITECTURE↑ ROOTING

↑MORPHOGENIC ABILITY↑↑ IN VITRO ESTABLISHMENT

VEGETATIVE GROWTH

Characterization Of Developmental PhasesCharacterization Of Developmental Phases

NEW STRATEGIES FOR IMPROVING HIGH-QUALITY PLANT PRODUCTION FROM MATURE

SELECTED TREES

REPRODUCTIVE MATURE

PHASE-CHANGE

VEGETATIVE MATURE

JUVENILE

EMBRYOGENIC

REINVIGORATION

MOLECULAR MARKERS (Physiological, Genetic Or Epigenetic Hallmarks) OF AGEING AND REINVIGORATION

UNDERSTANDING OF THE MOLECULAR BASIS OF AGEING AND REINVIGORATION

OPTIMIZATION OF BREEDING PROGRAMS

GENE REGULATION, EPIGENETICS

GENOMIC MAPINGGENE SEQUENCIATION

QTLs

GENE EXPRESSION

PROFILESPHYSIOLOGY

POLYAMINES AS PHYSIOLOGICAL MARKERS

Fraga M.F., Berdasco, M., Diego, L.B., Rodríguez, R yCañal, M.J. 2004. Changes of polyamine content associated with Pinus radiata D.Don and Prunus persica [L.]Bascth“Hakuho” ageing. Tree Physiology, en prensa.

Fraga M.F, Rodríguez, R. y Cañal M.J. 2003. Reinvigoration of Pinus radiata is associated with partial recovery of juvenile-like polyamine concentrations. Tree Physiology 23: 205-209.

Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. Phase-change related epigenetic and physiological changes in Pinus radiataD. Don. Planta 215: 672-678.

Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. In vitromorphogenic potential of differently aged Pinus Radiata D. Don. trees correlates with polyamines and DNA methylation levels. Plant Cell, Tissue and Organ Culture 70: 139-145.

AGEING

REINVIGORATION

PHASE-CHANGE

IN VITROBEHAVIOUR

EPIPHYSAGE Group

EPIGENETIC MARKERS, DNA METHYLATION

Fraga, M.F., Uriol, E., Diego L. B., Berdasco, M., Esteller, M., Cañal, M. J. y Rodriguez, R. 2002. High performance capillary electrophoretic method for the quantification of 5-methyl 2’-deoxycytidine in genomic DNA: Application to plant, animal and human cancer tissues. Electrophoresis 23: 1677-1681.

Fraga, M.F., Rodriguez, R. y Cañal, M. J. 2000. Rapid quantification of DNA methylation by high performance capillary electrophoresis. Electrophoresis 54 :2990-2994.

Fraga M.F, Rodríguez, R. y Cañal M.J. 2002. Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata. Tree Physiology 22: 813-816.

Fraga M.F., Cañal, M.J. y Rodríguez, R. 2002. Phase-change related epigenetic and physiological changes in Pinus radiata D. Don. Planta 215: 672-678.

QUANTIFICATION METHOD

QUANTIFICATION METHOD

AGEINGREINVIGORATION

PHASE-CHANGE

GENETIC MARKERS, RPT5 ATPase

Diego, L.B., Berdasco, M., Fraga M.F., Cañal, M.J., Rodríguez, R. y Castresana, C. 2004. A Pinus radiata AAA-ATPase, the expression of which increases with tree maduration. Journal of Experimental Botany 55 (402):1597-1599.

AGEING REINVIGORATION

EPIPHYSAGE Group

J APolyacrilamide

Gel

ds-cDNA

TTTTTTTTTTT-3´5´3´ 5´P PRIMER

5´5´3´

3´T PRIMER

AAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´

RNA-DNAhybrid

First strand synthesis(MMLV retrotranscriptase)

Reamplification of cDNAS (PCR)

AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´ AAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´

AAAAAAAAAA-3´5´

Juvenile Sample Adult SampleRNA isolation

(Dong & Dunstan, 1996)

DNAase I treatment

3 low stringency cycles (T. Anneal. = 40°C)

25 high stringency cycles(T. Anneal. = 60°C)

PCR

J APolyacrilamide

Gel

J APolyacrilamide

Gel

ds-cDNAds-cDNA


TTTTTTTTTTT-3´5´3´ 5´


5´5´3´

3´T PRIMER

5´5´3´

3´T PRIMER

5´5´3´

3´T PRIMER

AAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´

RNA-DNAhybrid


AAAAAAAAAA-3´5´ AAAAAAAAAA-3ÁAAAAAAAAA-3´5´TTTTTTTTTTT-3´3´ TTTTTTTTTTT-3´3´

RNA-DNAhybrid




AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´ AAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´

AAAAAAAAAA-3´5´


(Dong & Dunstan, 1996)AAAAAAAAAA-3´5´ AAAAAAAAAA-3ÁAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3ÁAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5ÁAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´

AAAAAAAAAA-3´5´ AAAAAAAAAA-3´5´


(Dong & Dunstan, 1996)

DNAase I treatmentDNAase I treatment



PCR



PCR

DIFFERENTIAL DISPLAY-RTPCR

F1 oripGEM®-T Easy Vector

(3015 pb) Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I

T7

SP6

+ cDNApGEM®-T

Easy

LIGATION

E.coli DH5competentes

TRANSFORMATION

LB-Amp

X-galSELECTIONMULTIPLICATIONMINIPREPS

AUTOMATICSEQUENCING


(3015 pb)


(3015 pb)pGEM®-T Easy Vector

(3015 pb) Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I

T7

SP6

Apa IAat IISph IBstZ INco IBstZ INot ISac IIEcoR ISpe IEcoR INot IBstZ IPst ISal INde ISac IBstX INsi I

T7T7

SP6SP6

+ cDNApGEM®-T

Easy

LIGATION+ cDNA

pGEM®-T Easy

LIGATION


TRANSFORMATION


TRANSFORMATION

LB-Amp

X-galSELECTION

LB-Amp

X-galSELECTIONMULTIPLICATIONMULTIPLICATIONMINIPREPSMINIPREPS

AUTOMATICSEQUENCINGAUTOMATICSEQUENCING

cDNA CLONING AND SEQUENCING

GENE EXPRESSION PROFILES IN

JUVENILE AND ADULT P. radiata

NEEDLES

PLANT MATERIAL

CANDIDATE GENES

pGEMT

cDNAs

cDNA INFERRED PROTEIN BEST ALINEATION

Pr β284 A1 Apoprotein, Photosystem I 93% Pinus thumbergii

Pr β286 P Glycoprotein 69% Arabidopsis thaliana

Pr β288 ATPase RPT5, 26S proteasome 95% Arabidopsis thaliana

Pr β381 Enoyl-ACP reductase 73% Nicotiana tabacum

Pr β382 Disulfide isomerase 75% Prunus avium

A P. radiata AAA-ATPase WHICH EXPRESSIONINCREASES WITH TREE MATURATION

Viveros Oihamberri, Vitoria (Spain).

Juvenile Trees SystemOpen pollinization

Selected Clones

Adult Trees SystemOpen pollinization

Selected Clones

PHASE-CHANGE VALIDATION

MACROGRAFTTEMPORALEVOLUTION

(1 month – 3 years)

CONSECUTIVEGRAFT

CONSECUTIVEGRAFT

NORMAL DEVELNON FLOWERING,CLONAL

MULTIPLICATION,VERY LOW RPT5 EXPRESSION

JUVENILE-LIKE

FLOWERING,LOW MORPHOGENIC

POTENTIALHIGH RPT5 EXPRESSION

ADULT FloweringBranches (A1)

FloweringBranches (A1)

EpicormicShoot (ES)

EpicormicShoot (ES)

Non FloweringBranches (A2)


REINVIGOR.


Selected Clones


Selected Clones



Selected Clones


Selected Clones


MACROGRAFTTEMPORALEVOLUTION

(1 month – 3 years)

CONSECUTIVEGRAFT

CONSECUTIVEGRAFT

NORMAL DEVELNORMAL DEVELNON FLOWERING,CLONAL


JUVENILE-LIKE





EpicormicShoot (ES)

EpicormicShoot (ES)



NON FLOWERING,CLONAL


JUVENILE-LIKE



ADULT



JUVENILE-LIKE



JUVENILE-LIKE



ADULT





EpicormicShoot (ES)

EpicormicShoot (ES)



REINVIGOR.

Diego et al. (2004) Journal of Experimental Botany 55(402):1597-1599

Cytokinin ResponsesCytokinin Responses

CRE1CK

ARR5

CYDC3

NIA1

Activator

GROWTH RESPONSES

RPN12a

Stress ResponsesStress Responses

REGULATORYPROTEOLYSISREGULATORYPROTEOLYSIS

Cell Cycle ControlCell Cycle Control

SEXUAL MATURATION(Physcomitrella,, Lilium)


TISSUEDIFFERENTIATION

(Zynnia)


(Zynnia)

Auxin ResponsesAuxin Responses

REPRESOR DEGRADATIONGENE EXPRESSION CONTROLREPRESOR DEGRADATION

GENE EXPRESSION CONTROL

Degradationof IAA /AUX

proteins

MORPHOGENIC ABILITIES


CRE1CK

ARR5

CYDC3

NIA1

Activator

GROWTH RESPONSES

RPN12a


CRE1CK

CRE1CK

ARR5

CYDC3

NIA1

Activator

GROWTH RESPONSES

RPN12aRPN12aRPN12a










(Zynnia)


(Zynnia)




(Zynnia)


(Zynnia)





proteins






proteins


26S PROTEASOME FUNCTIONS IN PLANTS

Proteasomicproteolysis

Auxin reception

IAA/AUXProteins

Messengers

Efectors

Primary Response

SHORT-TERM EFFECTS

SHORT-TERM EFFECTS

MIDDLE- AND LONG-TERM EFFECTS


RPT5

ES A1 A2

PROTEOLYTICCOMPETENCE

MODULATION?


MODULATION? Proteasomicproteolysis



Auxin reception

Auxin reception

IAA/AUXProteins

IAA/AUXProteins

IAA/AUXProteins

MessengersMessengers

Efectors

Primary Response

SHORT-TERM EFFECTS

SHORT-TERM EFFECTS



EfectorsEfectorsEfectors

Primary ResponsePrimary ResponsePrimary ResponsePrimary Response

SHORT-TERM EFFECTS

SHORT-TERM EFFECTS





RPT5

ES A1 A2


MODULATION?


MODULATION? RPT5

ES A1 A2


MODULATION?


MODULATION? RPT5

ES A1 A2

RPT5RPT5RPT5

ES A1 A2ES A1 A2


MODULATION?


MODULATION?

IS PROTEASOME MODULATING

CELLULAR COMPETENCE?

EXPRESSION LEVELS OF RPT5 homologue in Pinus radiata

Molecular marker of ageing and reinvigoration

Does it perform a role in competence modulation auxin-

dependent?

EPIGENETIC FACTORS

DNADNAiRNAs

5-

PROTEINSPROTEINSNON- HISTONES ALTERATIONS

HISTONES MODIFICATIONS:

- ACETYLATION

- METHYLATION

- PHOSPHORYLATION

-

DNA 5-

PROTEINS

-

-

-

-

-

WHICH INFLUENCES:TRANSCRIPTION,

REPLICATION,REPAIR

AND RECOMBINATION PROCESSES

METHYLCYTOSINE

UBIQUITINATION, ...

EPIGENETIC FACTORS

DNA iRNAs

PROTEINSNON HISTONES ALTERATIONS

HISTONES MODIFICATIONS:

ACETYLATION

METHYLATION

PHOSPHORYLATION

CONFORMATIONCHROMATIN

EPICORMICSHOOT

MATURE VEGETATIVE

MATURE REPRODUCTIVE

1-MONTH-OLD

NEEDLES (b1)

<1-MONTH-

OLDNEEDLES

(b0)

APICAL

BASAL

APICAL

BASALM

AT

UR

AT

ION

MA

TU

RA

TIO

N

MA

TU

RA

TIO

N

ONTOGENIC DEVELOPMENT




ARE HISTONE ACETYLATION LEVELS STABLE DURING AGEING AND REINVIGORATION? (If so, can AcH4 act as a molecular marker?)

MOLECULAR BASIS OF MATURATION AND DIFFERENTIATION

H4

HISTONE H4 TAIL MODIFICATIONS

HISTONE ACETYLATION DURING AGEING AND CHANGES DURING REINVIGORATION

DAPI AcH4

DAPI AcH4

ACETYLATED HISTONE H4

INMUNOLOCATION IN MATURE

VEGETATIVE (top) AND MATURE

REPRODUCTIVE (bottom) SHOOTS

DAPI + AcH4

DAPI

AcH4

ACETYLATED HYSTONE H4 INMUNOLOCATION IN

EPICORMIC SHOOT

LEVELS OF ACETYLATED H4 IN Pinus radiata NEEDLES ARE AGEING-DEPENDENT

GLOBAL HISTONE H4 ACETYLATION STRONGLY DECREASES WITH ONTOGENIC AGE.

REINVIGORATION COURSES WITH INCREASES IN ACETYLATED H4 LEVELS.

INMUNOLOCATION* Results are given as acetylation index (%).

1-MONTH-OLDNEEDLES (b1)

< 1-MONTH-OLDNEEDLES (b0)

APICAL BASAL APICAL BASAL

48

93 40 39

60 50 28 19

100

8 25 15

* Results are given as band intensity



34

34

34

34

34

34

* Results are given as acetylation index (%).




48

93 40 39

60 50 28 19

100

8 25 15




34

34

34

34

34

34





48

93 40 39

60 50 28 19

100

8 25 15




34

34

34

34

34

34





48

93 40 39

60 50 28 19

100

8 25 15








48

93 40 39

60 50 28 19

100

8 25 1548

93 40 39

60 50 28 19

100

8 25 15




34

34

34

34

34

34




INMUNOBLOTTING* Results are given as band intensity





1

0,77

0,34

0,63

0,30

0,23

106 kD80 kD

49,5 kD

32,5 Kd

27,5 kD18,5 kD

AMID

OBLACKST

ANDARS

EPICORM

IC SH

OOT

106 kD80 kD

49,5 kD

32,5 Kd

27,5 kD18,5 kD

AMID

OBLACKST

ANDARS

EPICORM

IC SH

OOT

MOLECULAR MARKER OF

AGEING

ESMVMR

STRUCTURAL ANALYSIS OF

BASAL PORTIONS OF 1-MONTH-OLD

NEEDLES

Top : MATURE VEGETATIVE

Bottom : MATURE REPRODUCTIVE

MERISTEM (HIPODERMIS)

MERISTEM (EPIDERMIS)

MESOPHYLL OF PARENCHYMA CELLS

MERISTEM (ENDODERMIS)

PERICYCLE

FLOEM

NOMARSKY

NOMARSKY

NOMARSKY

SIEVE PORES (CALLOSE

DEPOSITION)

REPLICATION

GLOBAL ACETYLATED H4 IS PREFERENTELLY CORRELATED WITH REPLICATION RATHER THAN WITH TRANSCRIPTION.

Jasencakova et al. (2000),The Plant Cell 12:2087-2100.

IN DIFFERENTIATED CELLS MOST OF THE GENOME CONSISTS OF HYPOACETYLATED INACTIVE CHROMATIN, WHICH MAY BE CONSIDERED THE “GROUND”STATE.

NOMARSKY

OPTIC MICROSCOPY

MERISTEM

EPICORMIC SHOOT

STRUCTURE

MERISTEMATIC CELLS IN EPICORMIC SHOOTS

CELL DIVISION

CELL MATURATION IN MATURE NEEDLES

GLOBAL H4 HYPOACETYLATION

GLOBAL ACETYLATED HISTONE H4 LEVELS

Molecular marker of ageing and reinvigoration

Molecular basis of organ maturation/

differentiation

HOW DOES DNA METHYLATION ACT IN THESE PROCESSES? DOES SPECIFIC METHYLATION MODULATE AGE-, PHASE-CHANGE- AND REINVIGORATION-RELATED EVENTS?

GLOBAL DNA METHYLATION (HPCE)MOLECULAR MARKER of:

-Ageing

- Reinvigoration

-Phase-change

BUT...

TFPROMOTER CODING

SEQUENCENON CODING

SEQUENCECODING

SEQUENCENON CODING

SEQUENCE MICROSATELLITES

CH3 CH3

TF

TFTFPROMOTER CODING

SEQUENCENON CODING

SEQUENCECODING

SEQUENCENON CODING

SEQUENCE MICROSATELLITESPROMOTER CODINGSEQUENCE

NON CODINGSEQUENCE

CODING SEQUENCE

NON CODINGSEQUENCE MICROSATELLITES

CH3CH3 CH3CH3

TFTFTF

TFPROMOTER CODING

SEQUENCENON CODING

SEQUENCECODING

SEQUENCENON CODING

SEQUENCE MICROSATELLITES

CH3 CH3

TF

TFTFPROMOTER CODING

SEQUENCENON CODING

SEQUENCECODING

SEQUENCENON CODING

SEQUENCE MICROSATELLITESPROMOTER CODINGSEQUENCE

NON CODINGSEQUENCE

CODING SEQUENCE

NON CODINGSEQUENCE MICROSATELLITES

CH3CH3 CH3CH3

TFTFTF SITE-SPECIFIC METHYLATION

WHAT KIND OF CELL ARE PREFERENTLY METHYLATED? DOES METHYLATION AFFECT MERISTEM AND/OR DIFFERENTIATED CELLS?

INMUNOLOCATION OF 5-mC

INMUNOLOCATION OF 5-METHYLCYTOSINE IN BASAL PORTIONS OF 1-MONTH-OLD P. radiata NEEDLES

Top : MATURE VEGETATIVE

Bottom : MATURE REPRODUCTIVE

DAPI

DAPI

5-mC

5-mC

OPTIMIZATION OF BISULFITE GENOMIC SEQUENCING IN PLANTS

-ALKALINE DENATURATION

- DEAMINATION

-DESULFONATION

-NEUTRALIZATION AND DESALTING

-AMPLIFICATION (PCR)

-CLONING AND SEQUENCING

DNAMethyl-easy

Mario F.FragaCecilia FerreroManel Esteller

DNAMethyl-easy

Mario F.FragaCecilia FerreroManel Esteller

CpG ISLAND

DO PROMOTER OR FIRST INTRON SEQUENCES CONTAIN

ANY METHYLATION-SUSCEPTIBLE ISLAND?

IS POLYAMINE BIOSYNTHESIS REGULATED BY SPECIFIC- METHYLATION?

ADC2

ARGININE DECARBOXYLASE EXPRESSION IN Arabidopsis thaliana

IS NOT REGULATED BY PROMOTER METHYLATION

ADC1POLYAMINE BIOSYNTHETIC PATHWAYS

OIHANBERRI S.A.

Ignacio GarcíaAitor Telleria

UNIVERSITAT AUTÒNOMA DE BARCELONA

Antonio TiburcioTeresa Altabella

UNIVERSIDAD DE OVIEDOEPIPHYSAGE GROUP (www.uniovi.es)

Roberto RodríguezMª Jesús CañalMaría BerdascoL. Borja DiegoRodrigo HasbunLuis ValledorCarlos Noceda

NEIKER

Enrique RitterSantiago EspinelAna Aragonés

CENTRO DE INVESTIGACIONES BIOLOGICAS.-C.S.I.C

Mª Carmen RisueñoPilar TestillanoPablo González- Melendi

CNIOMario F. FragaManel Esteller

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Presentación de PowerPoint · molecular markers (physiological, genetic or epigenetic hallmarks) of ageing and reinvigoration understanding of the molecular basis of ageing and reinvigoration