Physiological Respon of Tpd-affected Rubber Plant to Growth

Regulator and Antioxidant Treatments

Ade Fipriani

, Rosmayati

, Radite Tistama

and Revandy I. M. Damanik

Doctoral Program of Agricultural Sciences, Faculty of Agriculture, Universitas Sumatera Utara, Padang Bulan, Medan,

Indonesia

Program study of Agrotechnology, Faculty of Agriculture, Universitas Sumatera Utara, Padang Bulan, Medan, Indonesia

Sugei Putih Research Centre, Rubber Research Institute, Sungei Putih, Deli Serdang, Indonesia

Keywords: Bark, Leaf Fall Session, Physiological Characters and Protein.

Abstract: The research aimed to determine bark and latex the effect of applied NAA on thiol content, total protein and

peroxidase activity in the bark and latex of TPD –affected rubber tree (Hevea brasiliensis muell Arg.). The

research was conducted at the Sungei Putih Estate with 54 meters altitude, in leaf fall session from January

2018 to July 2018. The experiment was design using Split Plot Design, with three factors i.e: clones (RRIM

921 and PB 260), condition of plants (Healthy and TPD) and the formula (control, 2.5% and 5% the

stimulants). Parameter observed were thiol and protein content and peroxidase activity. The results showed

that the range of values bark and latex are Thiol 0.1-1.7 µM, Protein 31-124 ppm and Peroxidase 0,01- 0,9

unit/ppm protein, the clone of rubber plants was significantly affected the thiol’s. The NAA application

could improve the physiological characters of the rubber plant and the activities from all parameters

significantly affected by environmental conditions.

1 INTRODUCTION

Production of rubber plants with potential production

decreased reaching 114.74 kg / ha / t and 183.05 kg /

ha / year caused of Tapping Panel Dryness (TPD)

(Mochlisin & Tistama, 2014). In Indonesia, TPD

incident on productive rubber estate reach 20-50%,

which is characterized by the cessation of latex flow

and dryness for of the line of tapping. The economical

lossing caused by TPD incident are estimated to be

more than 1.7 billion/ year. Not balancing in latex

metabolism caused exploitation and the less

maintenance of plants are as mean factors inducing

TPD incident (Andryanto & Tistama 2014). Efforts to

limited incident are manuring, and restoration of

tapping fields for 6 months, with hormon application

and triadimephone 250 g/l (Sirait Syahnen, 2013).

However, TPD incident is still to be major problem in

Indonesian rubber plantations.

Physiological stress caused tapping in rubber tree

induces free radical compounds such as 0

-, H

OH*, QO* (Jacob, 1989). These reactive system

oxygen (ROS) enter into latex vessel cells (laticifer)

and interfere laticifer cell organels that are causing

cells disrupstion (Tistama, 2013). In TPD affected

plant, many genes related with a role in triggering

programmed cell death (PCD) were up regulated

during the early of TPD syndrome (Venkatachalam et

al. 2007).

Therefore, the earliest symptoms of histology

appearing in laticifer cells. So, it’s very likely to be

said that the trigger for a oxidative stress was known

in initially laticifer plants (Jacob and Krishnakumar,

2006). Joseph (2006) reported that the nutrient

content of P, Mg, and Mn in the latex is higher, while

Fe and Zn are lower in TPD-affected plant compared

to normal plants. Gebelin et al., (2013) said

detoxification of ROS by SOD, peroxidase and

catalase activity or through other non-enzymatic

mechanisms capable to detoxifying ROS from the

tissue without causing disrups. Thus, peroxidase and

catalase activities are key role in the process of

removing H

molecules in plant tissues.

Thiol in the plant tissue actives enzymes that have

control in respons to environmental stress conditions

as major antioxidace in the Hevea latex Zhang et al.

2017). Thiol levels are importance as indicatitor that

is related to the physiological susceptibility of latex,

especially in the incidence of TPD, and thiol status

shows of plants to the pressure of exploitation (Zhang

270

Fipriani, A., Rosmayati, ., Tistama, R. and Damanik, R.

Physiological Respon of Tpd-affected Rubber Plant to Growth Regulator and Antioxidant Treatments.

DOI: 10.5220/0008553002700274

In Proceedings of the International Conference on Natural Resources and Technology (ICONART 2019), pages 270-274

ISBN: 978-989-758-404-6

et al. 2017) The higher the intensity of exploitation

the lower the thiol level (Sumarmadji et al, 2004).

Sumarmadji and Tistama (2004) found thiol has the

ability to protect sub-cellular organelles and to cacth

the toxic oxygen molecules. This toxic oxygen

molecule causes exhaustion of latex vessel cells

which triggers TPD. The optimal content of thiol

ranges from 0.4 to 0.9 mM and the critical condition

is below 0.2 mm.

Peroxidase enzyme is one of the plant enzymes

that has a relationship with the process of resistance

(Zhang et al, 2017). According to Andrianto &

Tistama (2014) the difference in tolerance between

high and low metabolism of rubber clone is thought

to be due to the content of antioxidant compounds in

bark tissue and latex. To find out the sensitivity and

resistance of plants to disease attacks an approach is

used regarding the influence of environmental stress

on plant physiology processes. such as ethylene

treatment (Putranto et al. 2015).

Based on the early description, TPD is one of the

important problems that immediately need to be

resolved. The existence of leaf fall session can also

reduce the value of rubber production. Research on

rubber bark is needed cause the bark is where latex is

produced. Analysis of several physiological

parameters, is expected to be an early indicator of

healing process. These study aimed to increase the

cure rate of TPD by giving NAA formula and

Ascorbate acid and in the future it can reduce

production losses due to TPD and leaf fall session and

to examine changes in antioxidant compounds in

healthy and affected TPD plants with NAA and

ascorbic acid nutrition treatments.

2 MANUSCRIPT PREPARATION

2.1 Page Setup

Plant materials used for this study were PB 260 clones

(High metabolism) and RRIM 921 (Medium

metabolism) planted in 2006 with 3 x 6 m spacing.

The experiment was arranged with a Split-Split Plot

Design with three replications, each replication

consisting of 1 plant. The selection of the TPD

affected plants was carried out by cutting the tapping

panel with a tapping knife. Rubber plants are tapped

using a system tapping 1/2S d/3 (tapped every 3 days

with a length of 1/2 spiral of stem).

Formula for treatment in the form of minerals

follows the composition of Murashige & Skoog (MS)

with the addition of NAA (100 ppm, and ascorbic

acid (150 ppm). The solution was spryed on tapping

panel every 15 days for 3 month with a dose of 5 ml /

tree. Latex samples were collected into steril tube,

while bark samples were taken by cork borer

(diameter 1 cm) at 5 cm under tapping line, then

carrying in the box ice to laboratory.

2.2 Protein Extraction and Peroxidase

Activity

According to Bradford (1976), protein extraction for

lateks was perfomed by ten ml of each latex samples

were centrifuged at 15000 rpm for 20 min at

temperatur 4

C. Clear phase was transfered into the

new tube and used to protein and enzyme analysis.

Protein extraction for bark was performed following

of procedure Packeer-Mohamad et al. (2012). The

soft tissue of bark (3-4 mm from cambium) was

ground with liquid nitrogen and extracted in 0.2 M

phosphate buffer, pH 6.5, containing 0.25% (v/v)

Triton X-100 and 3% (w/v) polyvinylpyrrolidone

(PVPP). Bark samples were frozen in liquid nitrogen

and grinded in the mortar. One gram of bark powders

were extracted with phosphate bufer pH 7,2 using

vortex for five minutes. The extracts were centrifuged

at 12000 rpm in 4

C for 15 minute. Supernatan was

tranfered to new tube and ready for enzymatic

analysis. peroxidase activity uses phenol-

aminoantipirine A solution (phenol 810 mg solution

and amino antiphirine 25 mg in 50 ml water) and

solution B by mixing 30% H2O2 added with MES

Buffer solution pH 6 measured at 510 nm absorbance

(Shannon et al., 1996).

2.3 Physiological Analysis

For bark physiological analysis, one gram powder

was extracted in TCA 5% solution, then suspension

was centrifugated 10000 rpm for 15 minute. Analysis

of thiol content was based on the principle of its

reaction with dithiobis-nitrobenzoate acid (DTNB) to

form a yellow TNB absorbed at λ 421 nm

(nanometer) with a Beckman spectrophotometer DU

650 (Bobbiliof, 1923). Thiol indicated the level of

lutoids protection and the stability of latex. Thiol

contents were expressed in millimoles per liter of

latex (mmol.g

-1

2.4 Statistic Test

The results were tested by ANOVA and for the

treatment effect will be continued with Tukey

Multiple Comparison at alpha = 5%. Correlation

Physiological Respon of Tpd-affected Rubber Plant to Growth Regulator and Antioxidant Treatments

271

between parameters was tested Pearson Correlation

analysis using Minitab version 16 sofware.

3 RESULT AND DISCUSSION

3.1 Result

The latex thiol content in the both of rubber clone

(Figure 3) were lower in TPD affected plant than

health plants. PB 260 was higher thiol content both

in health or TPD partial than that of RRIM 921. In

leaf fall condition, latex thiol contents were 2-5 times

higher than full leaf condition. The thiol content was

decrease in health clones after the trees were treated

with ethepon. In TPD affected plants of PB 260, the

thiol content was categorized high and its was not

affected by ethepon treatments.In full leaf condition,

the thiol contents was normally in all of plant healthy

condition even in the plants treating with ethepon.

Generally, thiol content in bark of TPD affected

plant the RRIM 912 was higher than that of healthy

plants in March. The thiol content decrease in bark

of this clone after treating with ethepon 2,5% and

then increase with ethepon 5% treatment. In leaf

fall condition PB 260 has diference pattern in its

response to ethepon. Thiol bark content in May was

relatively flate by ethepon treatment.

3.2 Discussion

Latex POD activity was affected significantly by leaf

condition, ethepon treatment and clone. POD activity

in the bark PB 260 in full leaf periode was very high

in health plant, and the activity sharly reduced by

ethepone treatments. The bark high peroxidase

activity was negatively correlation with TPD level

(Tistama et al. 2019). The level of POD activity was

decrease by The health rubber tree could be indicated

by high POD activity in the latex and bark both in low

or high metabolism clone, however the level of POD

activity should be classified based on level of

physiology healthy. Opponent with POD activity, the

thiol content in March or in fall leaf condition. In May

when the leaf is full the thiol content decreased both

in the latex and bark. The thiol is dominan in thefall

leaf stress, while POD is dominan in ethephon

treatment.

(A)

(B)

Figure 1: Thiol content (mM) in health and TPD affected plant of the RRIM 912 and PB 260 latex (A= March and B = May).

(A)

(B)

Figure 2: Thiol content (mM) in the bark of health and TPD partial plant in March and May. (A= March and B = May).

ICONART 2019 - International Conference on Natural Resources and Technology

272

The content formulas shows the association of

formulas in improving of stress tolerance plants

through formulas supporting the performance of POD

in the detoxification process with both enzymatic and

non-enzymatic reactions. Although ethrel treatment

increases physiological stress in rubber plants due to

tissue injury which causes the sink and source process

to be unbalance. By combining ethrel and 100 ppm

and 150 ascorbic acid formulas as if to provide injury

and healing at the same time so that even though they

are still able to increase latex POD activity it is proven

by lower control plants in March observations with

the highest content below 0.3 units / ppm compared

to ethrel and formula which reached more than 0.4

units / ppm. This is presumably because of the

ascorbic acid content which acts as an antioxidant so

that it can support the role of peroxidase in the process

of removing ROS molecules such as H

. Winarsih

(2000) mention the main target of ROS is protein,

unsaturated fatty acids and lipoproteins, and DNA

elements including carbohydrates and RNA.

Unsaturated fatty acids are the most vulnerable to

ROS attacks. Ardiansyah et al. (2014) said ascorbic

acid functions as an antioxidant, cofactor enzyme and

as a cell signal modulator in a variety of important

physiological processes, including cell wall

biosynthesis, secondary metabolites and

phytohormones, stress tolerance, photoprotection,

cell division and growth.

The control plants there was an increase in the

content of POD which was very drastically observed

in May compared to March, this is thought to be

related to the deciduous process of leave fall session,

where May is the beginning of re-formation of

leaves so that the plants are no longer in stress.

Gunasekara et al., (2013) said that there were

differences in the production and response of each

clone to the seasonal pattern. The results showed that

PB260 clone activity looked more stable towards

changes in seasonal patterns compared to RRIM 912

clones which showed a very drastic difference. This

is in accordance with Siregar's statement (2016) PB

260 prioritizes thiol function in controlling stress

while in low metabolic plants prioritizes POD

enzymes. Siregar (2014) also said that canopy in

PB260 is generally thicker and thicker concentrating

on the number of fallers more and has a fast response

to changes in rainfall. This is related to the

hypersensitivity resistance gene that is dominantly

expressed in plants, the gene encoding peroxidee

enzyme and polyphenol peroxidase. Both of these

enzymes more commonly play a role in defense

mechanisms against diseases so that their activities

are used as resistance induction (Saravanan et al.,

2004).

Thiol contents in latex generally will increase if

the plant is exposed by TPD and given stimulants,

especially in PB260 plants. However, for healthy

plants the content will decrease. This happens

related to the level of exploitation and metabolism of

each clone. Research by Sumarmadji (2000) says

that the analysis of latex content in partial TPD

plants is known to show low thiol content even

though the plants were given stimulants ethepon and

Tistama et. Al (2006) which stated that thiol and

ATP in healthy plants were higher than those

attacked by TPD. This is thought to be an attempt by

the plant to overcome the conditions of oxidative

stress stress. According to Anggraini (2016), PB260

is more susceptible to attack by TPD.

Higher thiol content in PB260 bark compared to

RRIM 921 clones showed quick stater clones to

experience higher exploitation than slow stater. This

is an indication that slow stater plants are more

tolerant of TPD. The higher thiol content of the bark

can also indicate the presence of TPD whereas in

healthy plants the content of thiol bark is lower even

though it has been given stimulants up to 5% this is

because the plants give a better response to stimulants

combined with 100 ppm NAA formula and Ascorbic

Acid 150 ppm (Figure 4). Siregar et al., (2008)

reported that quick starter clones have a characteristic

that is the peak pattern of latex production occurs in

the early period, stimulant responsiveness,

susceptible to TPD and thin skin recovery, while slow

starter clones reach peak production in the middle of

tapping period, stimulant response, relatively

resistant to over-exploitation and thick skin recovery.

4 CONCLUSIONS

The Difference of antioxidant activities in the bark

and latex rubber plants (Hevea brasiliensis) change

according to environmental factors, clones, and

physiological disorders in this case because of the

provision of stimulants and nutrients and ascorbic

acid. Increasing the value of thiol can reduce the

value of protein and POD activity. Some parameters

that can be used as early indicators for detecting TPD

are bark POD activity and thiol latex value and based

on observations in the field of using 100 ppm NAA

formulas and Askorbit Acid 150 ppm can reduce the

level of TPD in rubber plants. From the results of this

study it is recommended to further investigate the

metabolism of rubber plant bark and its formulas in a

longer period of at least 6 months to 12 months.

Physiological Respon of Tpd-affected Rubber Plant to Growth Regulator and Antioxidant Treatments

273

ACKNOWLEDGEMENTS

These research was funded by the Ministry of

Research and Technology of Higher Education,

Lecturer Dissertation Research Scheme Fiscal Year

2018. Thank you also to all promoter and my family

for their moral support and all the friends who helped

this research.

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