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Discoveries in Agriculture and Food Sciences - Vol. 12, No. 6
Publication Date: December 25, 2024
DOI:10.14738/dafs.126.18013.
Rudiyanto, Y. F., Fanata, W. I. D., & Siswoyo, T. A. (2024). The Effect of Salinity Concentration on Proline Dehydrogenase (ProDH)
Gene Expression and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
Services for Science and Education – United Kingdom
The Effect of Salinity Concentration on Proline Dehydrogenase
(ProDH) Gene Expression and Proline Accumulation in Black Rice
and Red Rice
Yosefine Fetik Rudiyanto
Graduate School of Biotechnology, University of Jember.
Jl. Kalimantan No. 37, Jember 68121, East Java, Indonesia
Wahyu Indra Duwi Fanata
Graduate School of Biotechnology, University of Jember.
Jl. Kalimantan No. 37, Jember 68121, East Java, Indonesia
Tri Agus Siswoyo
Graduate School of Biotechnology, University of Jember.
Jl. Kalimantan No. 37, Jember 68121, East Java, Indonesia
ABSTRACT
The nutraceutical properties of the pigmented rice provide an opportunity to be
widely cultivated. A tendency to grow rice on sub-optimal land such as saline soil is
an alternative to support food security, but the adaptation under saline soil is still
questionable. This study was performed to determine the stress resistance of black
(var. Ketan Hitam), red (var. MS Pendek), and white rice (var. IR64) as a commercial
variety through proline accumulation and Proline dehydrogenase (ProDH)
expression under salinity stress and recovery. The results showed that salinity
stress increased the proline content in rice plants, with var. IR64 rice and var. MS
Pendek (red rice) accumulated the highest amounts of proline, and the expression
of the ProDH in IR64 and MS Pendek was increased in the recovery phase. These
results indicate the foundation for elucidating the mechanism response of black and
red rice to salinity stress and recovery ability.
Keywords: salinity stress, proline, gene expressions, ProDH
INTRODUCTION
Salinity stress is caused by the excessive accumulation of dissolved salts, which can significantly
impair plant growth and productivity. Salinity stress causes plants to experience osmotic stress,
accumulating osmoregulatory compounds, such as proline, which mitigate the effects of
reduced water potential within plant cells [1]. Osmotic adjustment is a critical mechanism that
enables plants to optimize water uptake from the environment, serving as a primary defense
strategy against dehydration [2]. The accumulation of proline under stress conditions is
facilitated by activating enzymes involved in proline biosynthesis, while enzymes responsible
for proline degradation are inhibited [3].
Proline dehydrogenase (ProDH) is an enzyme involved in proline degradation and is critical in
maintaining cellular homeostasis [4]. Under dehydrated conditions, proline works as an
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osmoregulatory compound, and in rehydrated conditions, it serves as an energy source through
its catabolism [5]. The equilibrium between proline synthesis and proline degradation, referred
to as proline homeostasis, is crucial for a plant's ability to withstand prolonged stress
conditions [6]. This homeostasis is integral in preserving redox balance, enabling plants to
resume growth and recover once the stress is alleviated [7].
Rice (Oryza sativa L.) is one of the most widely consumed cereal crops and a staple food for
many global populations. It is classified as a salt-sensitive species, particularly vulnerable
during the early vegetative and reproductive stages [8]. As reported by [9], the early vegetative
phase occurs 15-25 days after planting. Salinity stress with an electrical conductivity (EC) level
≥ 8 dS/m is considered moderate salinity stress for rice plants, while an EC ≥ 14 dS/m is
classified as high salinity stress [10]. According to [11], the salinity tolerance threshold for rice
is approximately 3 dS/m, with yield reductions reaching up to 12% for each additional dS/m
increase in salinity [12].
Black and red rice exhibit significant potential as functional foods due to their high
nutraceutical content [13], which is beneficial for health [14]. Therefore, it has the potential for
large-scale development. However, there is limited information regarding the resistance of
black and red rice varieties to salinity stress [15]. Assessing their resilience to such stressors,
including their capacity to recover once the stress is stopped, is essential. This study aims to
investigate the effects of salinity stress and recovery on proline content and ProDH gene
expression in three rice varieties: Ketan Hitam (black rice), MS Pendek (red rice), and IR64
(white rice, a commercial variety). By elucidating the recovery mechanisms of these plants
following stress exposure, the findings of this research are expected to contribute valuable
insights for the molecular development of salinity-resistant rice varieties, focusing on restoring
metabolic homeostasis in plants before stress exposure.
MATERIALS AND METHODS
Plant Materials and Experimental Conditions
This study was conducted at the Agrotechno-Park Jubung, the Integrated Laboratory University
of Jember, East Java, Indonesia. The rice seeds used in this experiment (IR64, MS Pendek, and
Ketan Hitam) were sourced from the UPA Laboratory, University of Jember. Homogenous and
healthy seeds were selected as plant material and cultivated under uniform conditions. Before
planting, the seeds were soaked in water for 24 hours and sown for 20 days, representing the
vegetative growth stage. Salinity treatment was simulated by applying NaCl to plants aged 21 –
28 days after planting (DAP). Three salinity levels were tested: 0 dS/m (control), 8 dS/m
(moderate), and 14 dS/m (high). NaCl was applied over a one-week period, with plant samples
collected at two distinct time points during the stress phase: 3 days after treatment (3 DAT)
and 7 days after treatment (7 DAT). Following the stress period, the plants were irrigated with
water to facilitate recovery. Recovery samples were then collected on day 1 and day 3 of the
recovery phase (1 DAR and 3 DAR). The experimental design used was completely randomized.
ProDH Expression Analysis
Total RNA was isolated using AccuZolTM a total RNA isolation reagent and approximately 1 μg of
RNA was used for first-strand cDNA synthesis with the AccuPower® CycleScriptTM RT Premix
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Rudiyanto, Y. F., Fanata, W. I. D., & Siswoyo, T. A. (2024). The Effect of Salinity Concentration on Proline Dehydrogenase (ProDH) Gene Expression
and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
URL: http://dx.doi.org/10.14738/dafs.126.18013
(dT20) solution reagent kit, following the manufacturer’s instructions. The cDNA product was
then amplified using PCR under the following conditions: 95°C for 5 min, 95°C for 30 s, 61°C for
30 s, 72°C for 1 min, and 72°C for 5 min as the final elongation. Primers used to amplify
antioxidant gene expression are listed in Table 1.
Table 1: Primer sequences for gene expression analysis
Gene Primer Sequence Molecular Weight (bp) References
Os-ProDH F: GGTTGGTCTTCCTTCAGGTGTGC
R: CATCAACATCATCAAACACCACTAT
106 [16]
Actin F: TCCATCTTGGCATCTCTCAG
R: GTACCCGCATCAGGCATCTG
335 [17]
Proline Content Analysis
Proline content was quantified using the method described by [18], and the results were
referenced against a pure proline standard curve, with units expressed as μg/mg. To prepare
the sample, a reactant mix comprised 1% ninhydrin, 60% acetic acid, 20% ethanol, and 19%
distilled water (aquadest). Approximately 50 mg of the sample was ground using a mortar, then
1 mL of a solvent mixture of 40% ethanol and 60% aquadest was added. The sample was then
transferred to an Eppendorf tube and incubated at 4°C for 24 hours. After incubation, the
sample was centrifuged at 10.000 rpm for 10 minutes. An aliquot of 200 μL of the supernatant
was combined with 800 μl of the reactant mix in an Eppendorf tube and heated at 95°C for 60
minutes. After cooling to room temperature, the sample was centrifuged at 2.500 rpm for 5
minutes. The final supernatant was transferred to a cuvette and analyzed using a
spectrophotometer at a wavelength of 520 nm.
Total Soluble Protein Content Analysis
Total soluble protein content was determined using the Bradford method [19], with Bovine
Serum Albumin (BSA) as the standard protein. A total of 0.3 g of sample was ground with a
mortar, then 900 μl of phosphate buffer was added to create a homogenate. The homogenate
was then transferred to an eppendorf and centrifuged at 10.000 rpm for 10 minutes to separate
the soluble protein in the supernatant from insoluble material. Subsequently, 5 μL of
supernatant was mixed with 45 μL of distilled water and 950 μL of Bradford reagent. The
mixture was vortexed thoroughly to ensure complete protein and dye interaction. The
absorbance value was measured at 595 nm using a spectrophotometer. The protein
concentration was determined by comparing the absorbance values to a standard curve
generated from known concentrations of BSA.
Total Chlorophyll Content Analysis
Total chlorophyll content was quantified using the spectrophotometric method [20]. A 0.5 g
rice leaf sample was collected and weighed. The leaf was then ground using a mortar until a
smooth homogenate was obtained. The ground leaf tissue was extracted with 1.5 mL of 100%
methanol and stirred to facilitate the release of chlorophyll from the tissue. The resulting
homogenate was transferred to an Eppendorf tube and centrifuged at 10.000 rpm for 10
minutes to separate the supernatant from the pellet. The supernatant was carefully collected
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and placed in a cuvette for spectrophotometric analysis. Absorbance was measured at three
wavelengths: 470 nm, 652 nm, and 665 nm to determine the total chlorophyll content.
RESULTS
Relative Expressions of ProDH
Molecular analysis was conducted to investigate the regulation of the ProDH in plants exposed
to salinity stress during the subsequent recovery phase. ProDH encodes the ProDH enzyme,
which plays a crucial role in proline degradation and is essential for maintaining cellular
homeostasis. The PCR products, shown as DNA bands in Figure 2, were generated using cDNA
as the template. The cDNA was synthesized through reverse transcription (RT) of messenger
RNA (mRNA) extracted from rice leaf samples. The expression of the ProDH in plants exposed
to salinity stress and recovery conditions is depicted in Figure 2.
The results indicated that ProDH expression was significantly elevated during the recovery
phase in all three rice varieties (IR64, MS Pendek, and Ketan Hitam), with similar intensities in
the DNA bands, as quantified using ImageJ. An interesting observation was found in MS Pendek,
where ProDH expression remained relatively constant under normal and recovery conditions.
Furthermore, MS Pendek exhibited a notably higher relative expression of ProDH during the
stress phase compared to the other varieties, IR64 and Ketan Hitam. The lowest expression of
ProDH in IR64 and Ketan Hitam differed. In IR64, the lowest ProDH expression was observed
under normal conditions, whereas in Ketan Hitam, it was observed during the stress phase.
C S R C S R C S R
Os- ProDH
Actin
IR64 Ms Pendek Ketan Hitam
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Rudiyanto, Y. F., Fanata, W. I. D., & Siswoyo, T. A. (2024). The Effect of Salinity Concentration on Proline Dehydrogenase (ProDH) Gene Expression
and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
URL: http://dx.doi.org/10.14738/dafs.126.18013
IR64 MS Pendek Ketan Hitam
0.0
0.5
1.0
1.5
Rice Varieties
Os-ProDH : Actin
Control Stress Recovery
Figure 2: Relative Expressions of ProDH
Proline Content
Proline content was analyzed under three levels of salinity: 0 dS/m (control), 8 dS/m (medium),
and 14 dS/m (high). Plants were exposed to these salinity treatments for 7 days, after which
the stress was terminated, initiating the recovery phase on the 8th day (1 DAR). Observations
continued until the 10th day (3 DAR). As shown in Figure 3, proline content increased in
response to salinity stress in all rice varieties. A decline in proline content was observed upon
cessation of salinity stress (recovery phase).
Figure 3: Proline Content in IR64, MS Pendek and Ketan Hitam under Salinity Stress and
Recovery
The highest proline content was observed on the seventh day of salinity stress, with the highest
salinity level was 14 dS/m. A similar peak in proline accumulation was recorded on the same
day at a salinity level of 8 dS/m; the levels were lower than those at 14 dS/m. During the
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recovery phase, a significant reduction in proline content was noted at both salinity (8 dS/m
and 14 dS/m). The decrease in proline content progresses, reaching the lowest levels on the
third recovery day. These findings suggest that proline accumulation is a key response to
salinity stress, while its subsequent decline during recovery indicates a return to normal
metabolic processes.
Total Soluble Protein Content
The results presented in Figure 3. indicate a decrease in total soluble protein content under
salinity stress conditions across all varieties, with both increasing salinity levels and prolonged
stress duration. Notably, the most significant reduction in total soluble protein content
occurred at a salinity level of 14 dS/m after 7 days of stress, showing a drastic percentage
decrease.
Figure 4: Total Soluble Protein Content in IR64, MS Pendek, and Ketan Hitam under Salinity
Stress and Recovery
During the recovery phase, an increase in the total soluble protein content was observed in each
variety. The highest percentage of total soluble protein occurred sequentially in the IR64, MS
Pendek, and Ketan Hitam varieties, with values of 54.06, 42.61, and 35.33%, respectively. These
results suggest that IR64 and MS Pendek exhibit faster recovery abilities than Ketan Hitam rice,
as indicated by the greater re-formation of total soluble proteins.
Total Chlorophyll Content
The effect of salinity stress on the percentage of relative changes in total chlorophyll content
(%) in rice plants is illustrated in Figure 5 below:
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Rudiyanto, Y. F., Fanata, W. I. D., & Siswoyo, T. A. (2024). The Effect of Salinity Concentration on Proline Dehydrogenase (ProDH) Gene Expression
and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
URL: http://dx.doi.org/10.14738/dafs.126.18013
Figure 5: Total Chlorophyll Protein Content in IR64, MS Pendek, and Ketan Hitam under Salinity
Stress and Recovery
Based on the research results in Figure 5, salinity stress significantly reduced the relative
percentage of changes in total chlorophyll content in rice plants. The most considerable
decrease in chlorophyll content occurred at a salinity level of 14 dS/m with a stress duration of
7 days across the IR64, MS Pendek, and Ketan Hitam varieties. However, all varieties showed
increased total chlorophyll content during the recovery phase. The increase in total chlorophyll
content from the highest stress level to the recovery phase was 66.81% for IR64, 49.75% for
MS Pendek, and 29.75% for Ketan Hitam.
DISCUSSION
The expression of a gene is indicated by the formation of mRNA [21], as reflected in the
thickness of the DNA band. A thicker DNA band signifies a higher quantity of mRNA produced,
which correlates with increased gene expression. As shown in Figure 2, the relative expression
of ProDH was up-regulated during the recovery phase, aligning with the role of ProDH in proline
catabolism once the stress is alleviated [22]. The breakdown of proline contributes to ATP
production through F1FO-ATPase [23], providing an essential energy source for plant regrowth
after the stress is removed [24]. These findings are consistent with the observed increase in
gene expression during recovery, highlighting ProDH’s role in facilitating energy production for
recovery processes.
The relative expression of ProDH was decreased and closely associated with proline
accumulation. However, high relative expression of ProDH was also observed under normal
conditions, particularly in MS Pendek. Under normal conditions, ProDH may be expressed as
part of the plant’s preparedness for potential stress [25]. This upregulation enables the plant
to respond more rapidly to stress before proline levels accumulate to harmful concentrations,
facilitating quicker adaptation to environmental challenges.
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Proline catabolism via ProDH is also linked to nitrogen metabolism in plants. Under normal
growth conditions, plants utilize various nitrogen sources, and proline degradation plays a
crucial role in nitrogen recycling [26]. ProDH activity in this context ensures the efficient use of
proline, a nitrogen-containing compound essential for plant growth and development. The
upregulation of ProDH under normal conditions reflects the need for a balanced proline
metabolism in plants [6]. ProDH regulates proline homeostasis, contributes to energy
production, and maintains proper nitrogen metabolism [27]. These functions underscore the
importance of ProDH in both stress responses and general plant growth and development.
Proline is an amino acid that plants synthesize as a protective mechanism against osmotic
stress. Proline acts as an osmoregulator, helping maintain the cells' osmotic balance. According
to [28], plants generally increase proline content in response to rising salinity levels, and this
content decreases once salinity stress is alleviated. Based on the graph in Figure 3, proline
content increased in each variety when exposed to salinity stress. Subsequently, proline levels
decreased when the salinity stress was removed during recovery.
The research results indicated that the highest proline content was observed at a salinity level
of 14 dS/m with a salinity application period of 7 days for the IR64, MS Pendek, and Ketan Hitam
varieties. This condition represents a state of severe stress for the plants, leading to a significant
accumulation of proline, which was higher compared to the salinity level of 8 dS/m and the
stress duration of 3 days. As stated by [29], plants typically increase proline content with higher
salinity levels and longer stress durations as a survival mechanism under abiotic stress
conditions. Proline content in IR64 and Ketan Hitam also increases under control conditions,
consistent with the findings of [30], who noted that plants can synthesize proline under stress
and non-stress conditions. Proline plays a crucial role in cell growth and differentiation, as it is
part of the protein cell wall component that functions in cell wall differentiation, plant
development, and stress tolerance [31].
Apart from being an osmoregulatory, proline is an important energy source, supporting various
metabolic processes necessary to continue growth and recovery in plants under stress [32].
According to [29], proline is no longer required as an osmoregulatory molecule under recovery
conditions. Excessive proline accumulation under optimal conditions can harm plants, as it may
lead to reactive oxygen species (ROS) formation. Therefore, proline must be rapidly catabolized
to prevent toxicity and allow the plant to resume its normal physiological processes and
complete its life cycle.
Proteins are composed of amino acids that play crucial roles in plant metabolism. Variations in
the total soluble protein content indicate amino acid synthesis in response to salinity stress
[33]. Under stressful conditions, plants break down proteins into free amino acids to help
maintain cellular osmotic pressure. According to [34], dehydration causes the cytoplasmic fluid
in the plant to become more viscous, leading to protein aggregation and denaturation. The
reduction in total soluble protein content in this study suggests that protein degradation during
the stress phase results in the formation of amino acids, particularly proline, which acts as an
osmoregulatory compound. This aligns with [35], who note that protein degradation yields free
amino acids, essential for maintaining cellular osmotic balance.
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Rudiyanto, Y. F., Fanata, W. I. D., & Siswoyo, T. A. (2024). The Effect of Salinity Concentration on Proline Dehydrogenase (ProDH) Gene Expression
and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
URL: http://dx.doi.org/10.14738/dafs.126.18013
Chlorophyll content, a key pigment component in plant leaves, is susceptible to salinity stress.
Under salinity stress conditions, the chlorophyll content in plant leaves is typically lower than
that of those growing in optimal environments. This decline in chlorophyll content under
salinity stress is commonly reported in various studies [36]. The study by [37] suggests that
excessive proline accumulation can have toxic effects, primarily by generating ROS, such as
hydrogen peroxide (H2O2), leading to lipid peroxidation and subsequent chlorophyll
degradation. This degradation is often manifested by a shift in leaf colour from green to
yellowish. Additionally, elevated salinity concentrations significantly reduce the rates of net
carbon fixation and adversely affect photosynthetic pigments, including carotenoids and
chlorophyll[38].
CONCLUSIONS
The relative expression of ProDH generally decreases under salinity stress. However, it is
upregulated during recovery, indicating its involvement in proline metabolism, specifically in
proline accumulation during stress and subsequent catabolism during recovery. Elevated
proline levels do not necessarily correlate with enhanced salinity tolerance; tolerance is
primarily determined by the plant's ability to recover after stress alleviation. This recovery
capacity is largely contingent upon the plant's ability to degrade proline and restore normal
physiological functions during recovery. In this regard, the rice varieties IR64 and MS Pendek
exhibited the highest levels of proline accumulation during the stress phase; however, they also
demonstrated a marked reduction in proline content during recovery, suggesting that these
varieties possess robust adaptive mechanisms in response to environmental stress. In contrast,
Ketan Hitam exhibits a higher salinity tolerance, as evidenced by its elevated ProDH expression
during the recovery phase and relatively low proline accumulation under stress conditions.
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and Proline Accumulation in Black Rice and Red Rice. Discoveries in Agriculture and Food Sciences, 12(6). 105-116.
URL: http://dx.doi.org/10.14738/dafs.126.18013
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