Indonesian Journal of Biotechnology

VOLUME 24(2), 2019, 88‐92 | RESEARCH ARTICLE

Analysis of ethylene biosynthesis gene expression profile during titanium

dioxide (TiO2 ) treatment to develop a new banana postharvest technology

Fenny M. Dwivany1,2,3,4,* Rizkita R. Esyanti1,3 , Veinardi Suendo5,6 , Aksarani ‘Sa Pratiwi1 , Annisa A. Putri1,3,4
1 School of Life Science and Technology, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, Indonesia
2 ForMIND Institute, Bandung 40132, Indonesia
3 Bali International Research Center for Banana, Universitas Udayana, Bukit Jimbaran, Kuta Selatan, Badung, Bali 80361, Indonesia
4 Bioscience and Biotechnology Research Center, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, Indonesia
5 Chemistry Department, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, Indonesia
6 Research Center for Nanoscience and Nanotechnology, Institut Teknologi Bandung, Jl. Ganesha No. 10, Bandung 40132, Indonesia

*Corresponding author: fenny@sith.itb.ac.id

SUBMITTED 17 Juny 2019 REVISED 23 August 2019 ACCEPTED 4 December 2019

ABSTRACT Banana is an important crop that demands proper methods in postharvest handling. As a climacteric fruit, the
banana fruit ripening process is affected by ethylene. Several methods have been developed to extend the shelf life of a
banana, such as using ethylene scrubbers. In this study, titanium dioxide (TiO2 ), a photocatalyst, was used as an alternative
method to delay the fruit ripening process. The effect of TiO2 on the ripening‐related gene MaACS1 was investigated.
Banana fruits were placed in a TiO2 ‐coated glass chamber and observed for ten days. Fruit ripening in the treated chamber
was delayed for eight days compared to the control. Total RNA was extracted from control and TiO2 ‐treated fruit pulp
and synthesized into cDNA. Reverse transcription PCR was performed to investigate the gene expression, which showed
that MaACS1 expression was relatively lower than treated control. The finding of these studies suggested that the TiO2
chamber has the potential to extend the shelf life of banana by delaying its ripening process and decreasing the expression
of MaACS1. To the best of our knowledge, no previous study has investigated the effect of TiO2 on the expression of genes
related to banana fruit ripening.

KEYWORDS banana; ripening; TiO2 ; postharvest; gene expression

1. Introduction is highly influenced by a gaseous phytohormone, ethy­

lene (C2 H4 ) (Guo and Ecker 2004). Ethylene is syn­
Banana is one of the most consumed fruits and catego­ thesized from S­adenosyl methinonine (AdoMet), which
rized as an important commodity in various countries. The is then converted into 1­aminocyclopropane­1 carboxylic
highly nutritious contents in banana such as vitamins A, acid (ACC) with the help of the enzyme ACC synthase
B1, B2, and C make the fruit popular around the world (Yang and Hoffman 1984). ACC is the intermediate pre­
(Cano et al. 1997). Indonesia is one of the top 10 countries cursor to generate ethylene. ACC is then converted into
in banana production (FAO 2019). However, Indonesia ethylene with the help of an ACC oxydase enzyme. Stud­
also faces problems in the postharvest handling of banana ies have demonstrated that MaACS1, a member of the
production. The majority of postharvest handling of ba­ ACC synthase (ACS) gene family, is responsible for the
nana is performed traditionally, which consequently leads ripening process of the fruit (Liu et al. 1999; Karmawan
to low­quality banana production. Some of the banana et al. 2009; Dwivany et al. 2016).
agroindustries around the world have attempted using a Titanium dioxide (TiO2 ) has been used as a photocata­
modified atmosphere and a controlled atmosphere tech­ lyst to degrade ethylene that is emitted by fruits at low tem­
nology to extend the shelf life of the fruit. However, such peratures (Hussain et al. 2010). Another recent study con­
techniques may not be effectively applicable, especially ducted using TiO2 nanofiber has demonstrated a delay in
for small Indonesian farmers in rural areas. Therefore, banana softening and color change (Zhu et al. 2019). It has
there is a need for other alternatives such as low energy been reported that ethylene degradation by TiO2 produces
technologies to extend the shelf life of banana by delaying carbon­dioxide and water and triggers a low ratio of O2 to
its ripening process. CO2 in the atmosphere (Charoenshap et al. 2012). These
Ripening is a natural process that occurs in fruits and conditions result in the reduction of ethylene biosynthe­

Indones J Biotechnol 24(2), 2019, 88‐92 | DOI 10.22146/ijbiotech.51718 Copyright © 2019 THE AUTHOR(S). This article is distributed under a
www.jurnal.ugm.ac.id/ijbiotech Creative Commons Attribution‐ShareAlike 4.0 International license.
Dwivany et al. Indonesian Journal of Biotechnology 24(2), 2019, 88‐92

sis since oxygen is needed to convert ACC to ethylene tal RNA was then synthesized into complementary DNA
by ACC oxidase (Kanellis et al. 2009). As mentioned (cDNA) using iScript™ cDNA Synthesis (Biorad cata­
above, ACC synthase is also an important enzyme which log number: 170­8890). Gene expression profile was
its activity also triggered by ethylene auto­catalytic reac­ analyzed by reverse transcription PCR (RT­PCR) using
tion. Thus, lower ethylene production will influence this the GreenTaq Master Mix kit (Promega, catalog num­
reaction (Inaba et al. 2007). In the present study, we in­ ber: M7122). All the PCR results were confirmed by
vestigated the effect of TiO2 applied as a thin coating on electrophoresis on 1% (w/v) agarose. Semiquantitative
the process of fruit ripening and the expression of the ba­ analysis of gene expression was performed using ImageJ
nana ripening­related ACC synthase gene, MaACS1. The quantification (http://www.imagej.net) as well as by T­test
results of this study demonstrated that TiO2 has the poten­ analysis.
tial to extend the shelf life of banana fruit.

3. Results and Discussion

2. Materials and Methods
3.1. Physical and Physiological Analysis
2.1. Fruit Storage Chamber Preparation Before conducting molecular analysis, the physical and
Fruit storage chambers made from glass (1.5 L) were used physiological characteristics of all bananas from the con­
in the experiment. The chambers were coated with a thin trol and treatment groups were evaluated (Dadzie and Or­
layer of TiO2 that was prepared by mixing crystal TiO2 chard 1997). Based on the ripening stage, the fruit can be
(J25) nanoparticles in ethanol as a solvent. The anatase divided into two categories, climacteric, and nonclimac­
crystal had a pH range of 3.5–4.5. The TiO2 nanoparticles teric (Friend and Rhodes 1981). Banana is a climacteric
were coated on the chambers using an airbrush sprayer and fruit and has the unique feature of ethylene production
then heated using a shot gun. All preparations were per­ during its ripening stage. During the entire ripening pro­
formed at the Chemistry Department, Institut Teknologi cess, the cells undergo several physical and physiological
Bandung. Charcoal pouches were placed in all chambers as well as molecular changes. Banana peel color changes
to absorb water produced from respiration. can be easily monitored by physical analysis before con­
Best quality Cavendish bananas were assorted, placed ducting physiological and molecular analyses.
inside the chambers, and stored until ripening for approx­ The assessments in this study included observation
imately ten days. The observation time points were cho­ of color changes in the banana peel, starch content
sen from day 0 to day 8 (D­day, D­1, D­3, D­6, and D­ analysis, pulp­to­peel ratio measurement, and TSS
8). Control group bananas were placed in a closed cham­ analysis. Color changes in the peel in both the control and
ber without light exposure, whereas treated group bananas treatment groups were observed during the ripening stage
were placed in a closed chamber with UV radiation (300– (Figure 1). Data were collected at the observation points
400 nm) for 24 h. Both treatments were performed at D­day, D­1, D­3, D­6, and D­8. Results showed that treat­
room temperature (26°C–27°C). The observation was per­ ment using the TiO2 chamber could delay the ripening pro­
formed at Bioscience and Biotechnology Research Center, cess as indicated by the color changes from mature green
Institut Teknologi Bandung. to yellow and brown. Banana peels in the control group
were generally brown in color since day 6, whereas the
2.2. Physical and Physiological Analysis treated banana peels were green to yellow in color.
Physical and physiological characteristics of the bananas Regarding starch content analysis, Dadzie and Or­
were evaluated based on peel color changes, starch con­ chard (1997) had stated that this method is simple, rapid,
tent measurements using the iodine test, pulp­to­peel ra­ and inexpensive to detect starch conversion into sugar dur­
tio measurements, and sugar content values measured us­ ing the ripening process visually. In this study, the treated
ing total soluble solids (TSSs) (Dadzie and Orchard 1997). bananas showed differences compared with the control
Peel discoloration and starch content measurements based group bananas, and after six days of observation, the pulp
on the iodine test during ripening were documented using still had the black coloration (Figure 1). This indicated
a Canon IXUS 230 HS digital camera. Meanwhile, TSS that the treated group had abundant starch content com­
measurements were performed using a hand refractometer pared with the control group. This result was also con­
(Atago™). firmed by the TSS values (Figure 2). Bananas from both
the control and treatment groups exhibited an increment
2.3. Gene Expression Profile Analysis in the o Brix during ripening. The faster ripening process
Molecular analysis was conducted to investigate the pat­ in the control group was also confirmed by its o Brix value
tern of MaACS1 gene expression in control and treated that was higher from D­day to D­6 than that in the treated
groups using gene­specific primers (Karmawan et al. group. However, on D­8, the sugar content in the con­
2009; Handayani and Dwivany 2014), wherein MaGA­ trol group was found to be lower than that in the treatment
PDH was used as a reference gene to compare the expres­ group. Regarding the color changes in the banana peel
sion profile. Total RNA was extracted as described by from green to yellow and brown after being overripened,
Cordeiro et al. (2008) with some modifications. The to­ studies have reported that these changes are caused by

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Dwivany et al. Indonesian Journal of Biotechnology 24(2), 2019, 88‐92

FIGURE 1 Color changes during ripening and the starch content analysis from control group [A] and treated group (exposed with UV light
and stored in FSC) [B] from each day of observation. The observation points were choosen between the day of observation to day 8 (D‐day,
D‐1, D‐3, D‐6, D‐8).

FIGURE 2 Total soluble solids of control and treated group from the FIGURE 3 Pulp to peel ratio from the control and treated groups.
pulp juice. The observation points were choosen between the day The observation points were choosen between the day of obser‐
of observation to day 8 (D‐day, D‐1, D‐3, D‐6, D‐8). vation to day 8 (D‐day, D‐1, D‐3, D‐6, D‐8).

the degradation of chlorophyll by the chlorophyllase en­ 1996). The brown spot or senescent spotting that appeared
zyme (Matile et al. 1996; Duan et al. 2007). Furthermore, on the ripe banana could be due to cell necrosis as a result
the yellow coloration on the peel is due to the increasing of chlorophyll degradation (Mosera et al. 2009). The color
amount of carotenoid pigments in the fruit (Subagio et al. and size of the spot could increase rapidly during the ripen­

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ing process (Karmawan et al. 2009). In addition, it has

been reported that starch contributes to 20% of the major
component in the fruit and gets converted into a carbon
source during ripening (Dettmann Bierhals et al. 2004).
During the process of ripening, starch conversion results
in a sweeter fruit. In this study, the starch content was as­
sessed qualitatively by the iodine test, in which iodine re­
acts with starch and results in black color around the fruit
flesh. On the other hand, the sugar content analysis done
by measuring the TSS in the pulp of the fruit indicated that
TSS values increased as the ripening process progressed
(Dadzie and Orchard 1997).
These results also corresponded to the pulp­to­peel ra­
tio measurement. Based on the data obtained, both the
control and treatment groups exhibited an increase in the
pulp­to­peel ratio during the ripening process (Figure 3).
However, the pulp­to­peel ratio has been considered to be FIGURE 4 Semi quantitative MaACS1 gene profile analysis. The
constant and better in assessing the ripening index (Dadzie observation points were choosen between the day of observation
and Orchard 1997). A change in the pulp­to­peel ratio is to day 8 (D‐day, D‐1, D‐3, D‐6, D‐8).
one of the physical indicators of ripening — sugar con­
centration increases in the pulp, which causes differences 4. Conclusions
in the osmotic pressure in the tissue. Meanwhile, the
peel loses its water content due to transpiration. Hence, This study has provided further information about the ef­
the pulp­to­peel ratio shows an increasing trend during fects of using the TiO2 chamber on the physical, physio­
the ripening process. This measurement can also be per­ logical, and gene expression changes during banana ripen­
formed using the peel­to­pulp ratio, but this would show ing. Earlier studies have also used TiO2 application to
a concomitantly decreasing trend with the ripening pro­ delay papaya and tomato ripening (Maneerat and Hayata
cess. In this study, bananas in the treated group exhibited 2006; Lourenço et al. 2017). However, to the best of our
a lower ratio than the control group bananas, indicating a knowledge, this study is the first investigation on banana
correlation with a slower ripening process in the treated ripening using the TiO2 chamber treatment, wherein the
group. results provided new insights into important gene expres­
sion changes occurring during ripening and further sug­
3.2. Gene Expression Profile Analysis gested that these changes can be used as an important
biomarker for evaluating banana ripening. The findings
The MaACS1 gene expression profile analysis was con­
of this study can provide a novel strategy to increase the
ducted as described by Handayani and Dwivany (2014) us­
postharvest quality of banana by prolonging its shelf life.
ing MaGAPDH as the reference gene (Figure 4). Results
Finally, these results can also provide a better understand­
showed that the expression level of MaACS1 in the con­
ing of the process of banana ripening and aid the develop­
trol group was statistically significantly higher than that in
ment of postharvest technology.
the treated group (p<0.05). Lower MaACS1 gene expres­
sion may result in lower ethylene synthesis, as the gene has
been reported to be a member of the ACC synthase gene Acknowledgments
family that converts AdoMet into ACC with the help of
the enzyme ACC synthase. As mentioned earlier, ACC is This research was partly funded by the Ministry of Re­
the intermediate precursor of ethylene and influenced by search, Technology, and Higher Education of the Repub­
ethylene auto­catalytic reaction. The change of O2 to CO2 lic of Indonesia. The authors thank Banana Group­Institut
ratio in the atmosphere by TiO2 treatment may result in a Teknologi Bandung for support during this study.
reduction of auto­catalytic reaction and ethylene biosyn­
thesis. The MaACS1 has been reported as a marker for Authors’ contributions
two different treatments to prolong banana fruit ripening,
such as low temperature and fungicide storage as well as FMD, RRE, and VS designed the study. ASP and AAP
chitosan coating treatment (Dwivany et al. 2016; Lustriane carried out the laboratory work, and FMD and AAP ana­
et al. 2018). lyzed the data and wrote the manuscript. All authors read
On the basis of previous studies, the expression level and approved the final version of the manuscript.
of the MaACS1 gene increases significantly during early
ripening and then decreases (Inaba et al. 2007; Karmawan Competing interests
et al. 2009). In this study, the expression of MaACS1 was
increased until day six and then decreased on day 8. The authors declare no competing interest.

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