The Cell and Molecular Physiology of the

Latex Producing Tissue, in Relation with

the Hevea Physiological Bark Diseases,
Stress and Latex Yield

Mahidol University, Institut de Recherche pour

Faculty of Science, le Développement (IRD),
Bangkok, Thailand Montpellier, France
Rubber tree
The 3 main limiting factors of latex yield,
at the level of the latex producing
tissues
The duration of the latex flow after tapping
: prolonged flow ---> more latex collected

The capability of latex cells to regenerate

their lost cytoplasm (latex) between two
consecutive tappings

Two types of bark diseases

(5-50% of the trees) :
– Tapping Panel Dryness: TPD
– Trunk Phloem Necrosis : TPN
 The early step of the 2 syndromes :
drying-up of the tapping cut

At this early step, discrimination between TPD and TPN, at

the level of the tapping cut, is almost impossible.
 Tapping Panel Dryness (TPD)

A physiological disease linked to over-exploitation

- Laticifers exhaustion (sugar depletion...?)

Gohet, 1996, etc.
Vichitcholchai et al., 1998

- Evidence of an oxidative stress, leading to latex cells

decompartmentation and in situ latex coagulation
Chrestin et al., 1983, 1989
Kongsawadworakul, 1997
Sookmark, 2002
TPD/TPN : Different syndromes
(physiological diseases)
young
scraped
TPD
TPD symptoms extends downwards
from the tapping cut (no necrosis)
= TPD (localized and “reversible”)

Inner necrotic symptoms, spreading

upwards from collar to tapping cut,
Invading the bottom part of the tree young
= TPN (“irreversible” phloem disease) scraped
TPN
 Development of TPN in Rubber Tree
scion

tapping panel

TPN is
• a physiological disease
of the inner phloem
• starting at the "scion-
rootstock" interface
• probably extending
through the circulatory
rootstock systems (phloem sieve
tubes and/or laticifers),
• in response to "severe
cumulative stresses" .
Nandris et al., 2004; Chrestin et al., 2004
 INTERNAL symptoms of BARK NECROSIS (young trees)

Early Symptoms :
after scraping of young "BN" trees,
the necrotic spots are always located spreading upward
at the collar (RS/S junction), to the tapping cut
the "final" step of the
Bark Diseases :
bark cracking
 Multifactorial causality of the disease

Release of Cyanide

Stresses

Climate
Exploitation
Clone
RS/S connection
Rhizogenesis
Compaction
Soil structure

• Combination of these multiple factors may vary from site to site (soil,
climate, exploitation system,...) and modulate the disease severity.

Nandris et al., 2004; 2005

 “To identify the molecular makers of stress and of
candidate genes for rubber yield and tree improvement ”

Molecular approaches:
Differential gene expression in the latex
and/or inner bark of rubber trees in various conditions

Techniques involving PCR amplification

 cDNA-AFLP
 Suppression Subtractive Hybridization (SSH),
 Validation of the candidate genes by sq- and/or qRT-PCR

Samples tested
- Diseased (TPD, TPN) / Healthy trees
- Ethrel® stimulated / Control trees
- High / Low yielding trees or clones
 Suppression Subtractive
Hybridization (SSH)

A/B

TPN / Healthy
Ethrel® stimulated / Control
High yield/Low yield
 Latex and bark sampling for RNA extraction

All samples were collected in

liquid nitrogen then stored at -80°C

Latex Sampling

Inner bark sampling

 Suppression Subtractive Hybridization (SSH)
Markers & CGs final validation :
RNA Northern blot, (sq-/q-) RT-PCR,…
N H

cDNA synthesis
PCR amplification
Suppression Subtractive
Hybridization
Spotting all unigenes on membranes
and macroarray analysis (hybridization)

Genes Classification
by functions
In common : 1
2

eliminated 3
4
5

SSH-N ESTs sequencing

SSH-H
SSH-N SSH-H Bioinformatic analysis
 Bioinformatic Analysis of our Inner Bark SSH
cDNA Libraries, from Healthy and Necrotic Trees
(Main Cell Function Classes)
Healthy (Healthy vs Necrotic) Necrotic (Necrotic vs Healthy)

Cell cycle Transport Cell cycle

1% Cell 2% Cell
Transport 7%
13% organization organization
signal
2% 10%
transduction
Signal Cell rescue 7%
transduction Cell rescue
6%
13% Secondary 13%
met
Secondary 6%
Gene
met Gene
Expression
7% Respiration expression
24%
3% 24%
Respiration
2%
Protein reg. Protein reg
primary met primary met
16% 19%
Healthy 16% 9%

Necrotic
The main function differences between the two SSH libraries : genes involved in :
cell rescue, cell organization, transporting systems and signal transduction…
 Macroarray Hybridization
(Zoomed part of a macroarray membrane)

The radioactive spots intensity is proportional to the corresponding gene

expression in the inner phloem of healthy or diseased trees.
The spots intensity is analyzed by the Array-Vision Software and
corrected for the local background, then standardized.
 Prevalidation by sq RT-PCR of the potential candidate TPN Markers

40 S
MW Neg N1 N2 N3 N4 N5 N6 N7 N8 N9 H1 H2 H3

Unknw1
MW Neg N1 N2 N3 N4 N5 N6 N7 N8 N9 H1 H2 H3

Unknw2
MW Neg N1 N2 N3 N4 N5 N6 N7 N8 N9 H1 H2 H3

SDRP

MW Neg N1 N2 N3 N4 N5 N6 N7 N8 N9 H1 H2 H3

TPN tree Healthy tree

Diagram of the symptoms displayed by young PB260 rubber
trees at a rather early step (3) of TPN development

Sampling for sq RT-PCR analysis

4

Drying area 3

Bark
sampling
areas

Inner necrosis
"Brown sheet "
2
Outer necrosis
* 1
 Expression of the SDRP and Unkwn1 & 2 genes at different levels of the
trunk of a TPN and a healthy tree (PB260) by sq RT-PCR (23-35 cycles)
N4 Unknw1 Unknw2 N4 Sdrp H4 UnKnw1
4
Unknw2
Sdrp
N3 N3 3 H3
*
23 26 29 32 35 23 26 29 32 35 23 26 29 32 35 23 26 29 32 35

N2 N2
H2

N1 N1
H1
2

* 1

Maximum SDRP expression at the front of the necrotic sheet expending to the cut
Maximum Unknw1 & 2 expression at both fronts of the expending necrotic sheet
 Suppression Subtractive
Hybridization (SSH)

A/B

TPN / Healthy
Ethrel® stimulated / Control
High yield/Low yield
Stimulation of Rubber yield with Ethrel®

300

Yield (% of control)
250

200

150

100

50

1 2 3 4 5 6 7 8 9
Tappings after stimulation

Application of Ethrel® (an ethylene releaser), at the level of

the tapping cut, induces a transient increase in rubber yield,
through activation of both latex flow and regeneration.
Study of the kinetic effects of Ethrel® on PB217 trees

Tapping (S/2)
PB217 trees (3 months unstimulated Exploitation S/2 d2
10 days untapped)

Stimulation 5% Ethrel Tappings / latex and bark collection

- 40 - 24 - 8 1st 2nd 3rd

Time before
the 1st tapping (h)
- 16 -4
 Suppression Subtractive Hybridization (SSH)
RNA

S C Stimulated vs Control trees

 Bark
cDNA synthesis
PCR Amplification
 Latex
Suppression Subtractive
Hybridization

Genes Classification
by functions
1

In common : 2
3

eliminated 4
5

SSH-S SSH-C
ESTs (~ 1,200/SSH) sequencing
SSH-S SSH-C Bioinformatic analysis (ESTDB/IRD)
 Expression analysis of some candidate genes from
stimulated latex and bark SSH libraries
Abscisic Acid Stress Ripening (ASR) Genes
90 Inner bark
80 Latex
70 BcASR2
LcASR2
60
BcASR3
50 ทิศตะวันออก
40S
40 BcASR4
ทิศตะวันตก
Treatment : C1 30C2 4 8 16 24 40h ทิศเหนือ
20 40S
C1, C2 : Control 10 Treatment : C1 C2 4 8 16 24 40h
4, 8, 16, 24, 40h : hours after stimulation
0
ชวงที่ 1 ชวงที่ 2 ชวงที่ 3 ชวงที่ 4

All bark and latex ASR isoforms were transiently down-regulated by

Ethrel® stimulation.
 Abscisic Acid Stress Ripening (ASR) Genes

ASR genes are known to be regulated by stress and different

hormones or phytochemicals (ABA, MeJA), but no data have ever
been reported on their regulation by ethylene.

Recent study reported that ASR protein could bind to the promoter
of hexose transporter gene suggesting the proposed role of ASR as
a trancription factor involved in sugar uptake.

Çakir et al., 2003

 Suppression Subtractive
Hybridization (SSH)

A/B

TPN / Healthy
Ethrel® stimulated / Control
High yield/Low yield
 Suppression Subtractive Hybridization (SSH)
RNA

H L High yield vs Low yield

 Bark
cDNA synthesis
PCR Amplification
 Latex
Suppression Subtractive
Hybridization

Genes Classification
by functions
1

In common : 2
3

eliminated 4
5

SSH-H SSH-L
ESTs (~ 1,200/SSH) sequencing
SSH-H SSH-L Bioinformatic analysis (ESTDB/IRD)
Looking for Expression Markers of Stress and Yield
Candidate Genes

Collaborations
 We have started in May 2005 a common IRD-MU-KKU experiment in
the Khon Kaen area, with ecophysiologists, to study the expression
of some genes in response to environmental stress (soil and drought).
 Bark and latex collection at various time along the seasons.

 We have started since 2005 a collaboration with PIAF-UMR

(Clermont-Ferrand / Prof. S. Sakr), under the Franco-Thai Research
and Education Program.
A "specific" approach : molecular characterization of the water and
sugar transporters in the rubber tree laticifers and inner bark tissues.
(PhD : K. Tungngoen and A. Dussotoit)
 Looking for Expression Markers of Stress and Yield
Candidate Genes
Collaborations
 We have started in May 2007 a common CIRAD (A. Leconte) -RRIT-IRD-
MU experiment in the CRRC (P. Chantuma and P. Romruensukharom) in a
BPM24 field, attempting to induce (or avoid) bark diseases (TPN, TPD)
through various treatments and tapping systems, starting at the trees opening
 Monthly LD, yield and health control, as well as collection of bark
and latex for protein/nitrogen storing and molecular biology studies
 We could show that, at the very early step of the TPN induction, the
previously evidenced TPN markers were expressed and some plasma
membrane aquaporins sharply down-regulated (water stress?)

 MU-IRD would like to have further collaboration with CIRAD and RRIT for :
 Mapping of latex and phloem expressed genes, including our candidate
genes related to stress and yield (K. Prapan / RRIT PhD student).
 Dr. Panida Somjintana Ruderman
Dr. Hervé Chrestin Kongsawadworakul Kessarin Tungngoen Manassawee
Yield Marker, RRIT Kullaya Prapan Lertpanyasampatha
IRD-MU Aquaporins, MU
Plant Science, MU Marker Mapping, RRIT TPN, MU

Assoc.Prof. Jarunya
Narangajavana Dr. Unchera Viboonjun
Chutima Charoenwut Sirawan Chatsapsin
Plant Science, MU Stress Genes, MU Tiwa Rotchanapreeda
Biotechnology, MU Yield Marker, MU
Yield Marker, MU Pattharapong Inchana
Lipoxygenase, MU

Yaowalak Mangpung Alitra Tamma

Yield Marker, MU Stress Genes, MU
Paweena Chuenwarin
Jean-Philippe Pichaut
Benjamas Towaranonte Wipada Polsri Yield Marker, MU
VIE/IRD
Yield Marker, MU Yield Marker, MU
Ornwatsa Pueksuk
Lipoxygease, MU