Effects of Prohydrojasmon on Growth and Secondary Metabolism in Leafy Vegetables

概要

Appended Form No. 3(Doctor)

Ab stract of Th esis
Affiliation

Doctoral Program in

Life Science Innovation
: Food Innovation

Degree Programs in Life and Earth Sciences
Graduate School of Science and Technology
Student ID number

202230321

N

Haidar Rafid Azis

a

m

e

Thesis Title

Effects of Prohydrojasmon on Growth and Secondary Metabolism
in Leafy Vegetables

Japanese Title

（プロヒドロジャスモンが葉物作物の生育および二次代謝に及ぼす
効果）

Abstract of Thesis
In the last decade, there is ever growing number of non-communicable disease, such as
cardiovascular disease, obesity, neurogenerative disease, and other deadly diseases. In parallel to that,
awareness towards healthier diet and lifestyle also increasing to some degree. Several secondary
compounds from plant origin gained more attention primarily due to its health-promotion properties.
Epidemiologically, high intake of food rich in phenolic compounds such as polyphenols have been
strongly associated with the diminish risk of various disease and improve overall health. Consequently,
the demand towards these kinds of foods also rising substantially. On the other hand, nowadays plants
facing more intense environmental challenge amidst the surge of climate change. In such circumstance,
numerous processes in the growth and development of plant become compromised physiologically. This,
to the greater extent can negatively impact the productivity and yield of the crops, and ultimately can
lead to economical loss. To certain extent, plant growth regulator (PGR) may promisingly come up as a
solution to tackle these two problems in the society.
PGRs are substances of natural or synthetic origin that is used mostly in a miniscule amount to
regulate certain physiological and metabolic processes in plant. The term PGR often interchangeably
used with the plant hormone, which by definition has more nuance to the natural origin substances but
has similar role in plant. Basically, PGR does not have nutritive value, but it has critical role in regulating
multitude processes in plant, ranging from development of flower and fruit, maturation, up to senescence,
and many other processes. Jasmonic acid (JA) and its derivatives have attracted interest among the main
plant hormones. It is relatively new and considerably less explored compared to the classic hormones
that have been discovered decades earlier. JA is known primarily for its function in plant defense and
stress response. It also numerously reported having a significance effect on promoting the secondary
metabolites production in various plant crops.
New types of plant growth regulator analogous to jasmonic acid namely prohydrojasmon (PDJ)
was created and developed just recently. PDJ is regarded as a jasmonic acid-like synthetic compound
that can also stimulate the biosynthesis of secondary metabolites in crops. However, at the same time,
PDJ might also possibly interfere with the growth of the plant, particularly when applied at higher
concentration. To date, most studies on PDJ utilization are still highly concentrated on fruits, particularly
related to fruit ripening and coloration, and few others to the stress response. PDJ utilization on
vegetables is still lacking and considered less explored. Given that PDJ reportedly improved some
phytochemicals in fruits, it is plausible to expect that PDJ may also enhance the production of some
functional compounds in vegetable crops easily and safely.
The objective of this research is to carefully examine and consider the prospective application
of PDJ on vegetable crops by conducting experiments to measure their impact on growth and secondary
1

metabolisms. PDJ has potential inhibitory effect on the growth which is important to note. But on the
other hand, it may help to promote secondary metabolites production in crops. It is important to find out
the “sweet spot” between the negative and positive effect of PDJ, particularly here in vegetable crops.
By identifying and balancing these two opposing aspects, it is expected that the optimum PDJ utilization
on vegetables be obtained. Further, this research also attempts to elucidate the mechanism action of PDJ
on these two aspects. Hopefully, the information gathered from this research can be useful to help
develop a better agricultural system, and particularly to offer an alternative approach to produce a highquality vegetable with higher content of secondary compounds that beneficial to improve human health.
To accomplish the objective, this research is structured into five chapters. The first chapter explained
the general introduction that consist of the research background, related literature reviews, objectives of
the research and composition of doctoral thesis. Chapter two investigated PDJ effect on the growth of
plant in normal and spindly growth seedling setting using komatsuna and eggplant. Chapter three
covered the investigation of PDJ effect on secondary metabolism, particularly it assesses PDJ effect on
the phenolics accumulation in komatsuna and lettuce along with their antioxidant activity. Chapter four
elucidated the mechanism action of PDJ effect on the on the growth of plant, specifically on komatsuna.
Lastly, chapter five provides the general discussion to wrap up and summarize all findings and finally
conclude the research.
Komatsuna and eggplant were grown in soil at lab-scale using growth chamber to investigate
the effect of PDJ on growth. At certain stage of growth, these vegetable crops were treated with PDJ at
various concentrations (200, 400, 600, and 1000 ppm v/v), which were applied as a foliar spray and
drip-wise method. According to the findings, PDJ had no significant inhibitory or stimulatory effect on
the growth of aerial parts in either plant. Lower concentrations of PDJ also had no significant inhibitory
effect on the roots of either plant. However, at higher concentrations, PDJ started to suppress the root
growth, particularly it reduced the length and weight of root in both plants. PDJ at higher concentration
also was found to cause a wilting leaves phenomenon in komatsuna. This phenomenon along with the
yellowing leaves are thought to be associated with the promotion of senescence. Eventually, this gives
important clue to elucidate possible mechanism action of PDJ effect on the growth, which is investigated
further in the latter chapter.
Although the inhibitory effect of PDJ at high concentration is seemingly unfavorable, but it
might be useful to overcome the spindly growth seedling, a phenomenon that sometimes occur and can
lead to the poor seedling establishment. Spindly growth occurs in a plant grown under dark and/or dimlit conditions. It is characterized by the long hypocotyl, small and closed cotyledon, and short root.
Spindly growth seedling is considered unfavorable because it can lead to low productivity and yield.
Given that PDJ at higher concentration indicates a strong inhibitory effect, the possibility of PDJ to be
used for controlling etiolation in seedlings was investigated.
Here, komatsuna and eggplant were cultivated in an almost similar fashion, except they were grown in
low light conditions (PPFD: ca. 15 μmol m−2 s−1) to allow hypocotyl elongation. PDJ was prepared
only at concentrations of 400 and 600 ppm which is regarded as a rather higher concentration. Likewise,
PDJ was applied as a foliar spray and drip-wise method. The results suggested that PDJ substantially
reduced the elongated hypocotyl of both plants. It also implicates the root growth, and weight of
seedlings. All of these indicate that PDJ can be used to control the spindly growth in seedlings.
Additionally, it was observed that PDJ applied as spray showed stronger inhibition compared to PDJ
applied as drip-wise, and different plant species showed different sensitivity towards PDJ exposure.
Investigation of total phenolic and anthocyanin content, and antioxidant activity were carried
out on the PDJ-treated komatsuna and lettuce to measure its effect on secondary metabolism in these
vegetable crops. Soil and hydroponic methods were used to grow komatsuna and lettuce, respectively.
Both plants were sprayed with PDJ at varying concentrations (0.03, 0.13, 0.25, 50, and 100 ppm).
Contents of total polyphenols and color metric absorbance of methanol extract from komatsuna and
lettuce showed that PDJ significantly induced total phenolic and anthocyanin content in both plants with
an increase of up to 15%. Additionally, PDJ also significantly increased antioxidant activity in lettuce.
While insignificant in komatsuna, PDJ still shows increased antioxidant activity tendency. Conclusively,
it is revealed that PDJ implicates secondary metabolism in vegetable indicated by the significant increase
of some phenolic compounds along with the increase in antioxidant activity. It is also confirmed that
PDJ utilization is not restricted to the fruit, but it also applicable to the vegetable crops.
To help elucidate the mechanism action of PDJ on secondary metabolism, our recent study on
2

red-leaf lettuce was used as a reference. Metabolomic analysis of the study identified accumulation of
some phenolic and anthocyanin compounds in the lettuce leaves treated with PDJ.
The accumulating phenolics were subsequently identified by HPLC and LC-MS studies as
caffeoylmalic acid, caffeoyltartaric acid, dicaffeoylquinic acid, chlorogenic acid, and chicoric acid,
which are all caffeic acid derivatives. Meanwhile, the accumulating anthocyanins were identified as
cyanidin-3-O-glucoside (Cy3G) cluster, along with its malonylated form (Cy3MG), and also further the
methylated form of malonyl glucoside (Cy3MG-Me). Further, upregulation of PAL attributable to the
phenolics accumulation, and increase F3H, and ANS expression owing to the anthocyanin accumulation
was indicated by gene expression study. Mechanism action of PDJ on secondary metabolism might be
explained by the change expression of these genes. ...