AJCS 15(01):107-113 (2021)
doi: 10.21475/ajcs.21.15.01.2889

ISSN:1835-2707

Characterizing the photosynthetic ability of the submergence-tolerant rice varietyof Inpari30 via maximum quantum yield performance during transient flooding
stress and recovery
Arinal Haq Izzawati Nurrahma1, Shin Yabuta2, Ahmad Junaedi3, Jun-Ichi Sakagami2*
1

The United Graduate School in Agriculture Sciences, Kagoshima University, Kagoshima, Japan
Faculty of Agriculture, Kagoshima University, Kagoshima, Japan
3
Faculty of Agriculture, Bogor Agricultural University, Bogor, Indonesia
2*Corresponding author: sakagami@agri.kagoshima-u.ac.jp
Abstract
Submergence is an environmental challenge for crop cultivation which causes physiological perturbation and yield loss. Tolerant
genotypes are characterized by the ability to maintain physiological processes, especially photosynthesis, minimizing the negative
effects of flooding stress. This experiment was aimed to examine the photosynthetic ability during submergence and a recovery
period in rice variety of Inpari30 (carrying gene Sub1) compared to IR72442 (an elongation type) under control and submergencetreatment. A randomized complete block design was employed in this experiment with three replications. The first factor was theenvironmental condition consisting of control and submergence. The second factor was rice varieties consisting of Inpari30 and
IR72442. Fourteen-day-old seedlings were submerged under 35 cm of water depth for 6 days and re-aerated by lowering the waterlevel up to the stem base for 6 days of recovery. Measurement of plant height, SPAD, Fv/Fm and photosynthetic rate were taken
continuously from the same plant sample following the experimental stage. The results showed that shoot length increased
significantly more in IR72442 than in the quiescent Inpari30. The noticeable decline was observed in the photosynthetic rate ofboth varieties during submergence with chlorophyll content and chlorophyll fluorescence (Fv/Fm) decreased more severely inIR72442 than in Inpari30. After flooding, Inpari30 adapted quickly to the aerobic environment, as shown by a recovery in Fv/Fm andaccumulated dry weight more quickly than IR72442. We concluded that the Sub1 genotype of Inpari30 confers the ability tomaintain maximum quantum yield of PSII under conditions of limiting gas exchange for photosynthesis for adapting post
submergence.
Keywords: Sub1A, elongation, Fv/Fm, rice submergence, flash flooding.
Abbreviations: DAS_days after seeding.
Introduction
Complete submergence is defined as a condition when theentire plant is fully and continuously submerged underwaterfor 7–14 days (Das et al., 2005; Nishiuchi et al., 2012).
Submergence limits plant growth through slow diffusion
because the rate of oxygen diffusion is reduced underwater(Amstrong, 1979; Jackson et al., 1984), which inhibits
metabolic processes (Setter et al., 1989). A transient
flooding event perturbs plant metabolism twice: once during
submergence and again during the re-aeration that occurs
after the water recedes triggering internal stresses that
affect plant growth and survival (Pradhan and Mohanty,2013). When a plant becomes submerged, the environment
becomes limited to gas diffusion and low light intensity,
which together result in oxygen deficiency, decreasedtranspiration, and starvation. Upon re-aeration, the plant is
exposed to a sudden excess of oxygen and relatively highlight intensity, which leads to oxidative stress anddehydration (Panda and Sarkar, 2012; Tamang and Fukao,2015).

Any sudden decrease in gas diffusion is a major problem forplants, limiting the photosynthesis and respiration processes
that require oxygen and carbon dioxide as substrates(Pradhan and Mohanty, 2013). Among photosynthesis
variants, photosystem II (PSII) is known to be the mostsensitive to environmental changes, including submergence
before irreversible damage becomes apparent (Sayed,2003). Damage to PSII can be detected by observing thepotential maximum quantum yield (proportional to the
Fv/Fm ratio, a measure of chlorophyll fluorescence), which
generally decreases in plants suffering environmental stress.
In general, a decline in Fv/Fm is an expression of aphotoinhibition phenomenon (Björkman and Demmig, 1987)
involving not only photodamage to the photosynthetic
system, but also acting as a photoprotective mechanism(Krause and Weis, 1991). The chlorophyll fluorescenceparameter Fv/Fm has been widely used for measurement of
maximum quantum yield of PS II, as it is a non-invasive
technique suitable for studying the tolerance of thephotosynthetic apparatus to environmental constraints (Van107

Kooten and Snel, 1990; Murchie and Lawson, 2013).
Fluorescence emission is indicative of the composition of theantenna system used in photosynthesis, which is influenced
by chlorophyll content per leaf area (Lichtenthaler et al.,1986). Chlorophyll fluorescence induction provides
information on almost all aspects of photosynthesis in PSII
and the response of plants to environmental factors such as
stress and nutrient availability (Björkman and Demmig,
1987; Krause and Weis, 1991; Zhu et al., 2005). Threefunctions of chlorophyll are important for plant growthduring photosynthesis: light absorbance, transfer of the lightenergy by resonance energy transfer into a specificchlorophyll pair in the reaction centre of the photosystem,
and facilitation of the charge separation leading tobiosynthesis (Eugene and Govindjee, 1965; Murchie andLawson, 2013).
Rice variety of Ciherang is widely grown by the farmer inlowland rice field in Indonesia which sensitive to unexpected
inundation (Nugraha et al., 2017; Nurrahma et al., 2017). As
the country where rice stands as the main staple food,providing suitable rice variety that able to maintain highyield after exposed by flooding due to uncertainties rainfall
is mandatory. It will also promote an approach to increase
productivity and intensify of rice production in the lessfavourable environment, i.e. tidal lowland or coastal which
also prone toward submergence (Rumanti et al., 2018).
A major quantitative trait locus (QTL) responsible forsubmergence tolerance, Sub1, has been widely studied in
FR13A rice (Oryza sativa L.) and regarded as conferring
submergence tolerance in rice breeding (Xu and Mackill,
1996; Xu et al., 2006; Panda and Sarkar, 2012). For example,
the development of submergence tolerance throughintrogression of Sub1 into the high yielding rice variety
Inpari30 in Indonesia recently resulted in no significant
difference from the parental variety (Ciherang) in all traits
observed under normal conditions (Septiningsih et al., 2015;
Nugraha et al., 2017). However, limited study reported
photosynthetic activity following submergence which
responsible during recovery and gain assimilates for yieldproduction to characterize newly developed submergence
tolerant variety of Inpari30.
The effect of submergence to shoot elongation was tested to
15 genotypes of Oryza sativa for 7 days under 80 cm water
depth resulted the highest ratio of submerged to control
plant in IR72442 among all cultivar. Then, this variety used
as a typical shoot-elongating cultivar to evaluate thedifference of chlorophyll content and chlorophyll
fluorescence under submergence compared to non-shootelongating cultivar (Sone et al., 2011). The result concluded
that shoot-elongating cultivar was able to maintain thephotosynthetic capacity of the newly developed leaf during
submergence by prompt reduction of chlorophyll andchlorophyll fluorescence in the leaf that developed before
submergence.
In this experiment, we would like to evaluate photosynthetic
performance of Inpari30 during submergence following by
subsequent reaeration, as a basic understanding to explain
the physiological mechanism of Inpari30 overcome
submergence stress. Besides, we also aimed to clarify thedifference photosynthetic performance between non-shootelongation type of Inpari30 and shoot elongation type ofIR72442 by integrating measurements from gas exchange
analysis, chlorophyll content (SPAD value), and chlorophyll
fluorescence (Fv/Fm).

Results
Effect of submergence on the increase of plant length
Plant length of submerged IR72442 increased significantly
more than its control (>34.3% after 6 days of submergenceand >40.9% by the end of the 6-day recovery period) andsignificantly more than both the submerged and control of
Inpari30. Conversely, the smaller increase in plant length ofsubmerged Inpari30 was not statistically significant
compared to its control. The ratio of plant lengths (aftersubmergence versus control) was 1.23 for IR72442 and1.02 for Inpari30 (Fig. 1). The slow growth characteristic
under submergence conferred by Sub1A was noticeable in
the limited change in plant length of Inpari30.
Submergence decreased chlorophyll content (SPAD value)
The SPAD values for IR72442 decreased significantly versus
control (72.4%) after submergence whereas those for
Inpari30 decreased by only 14.4% (not significant). The valueof all treatment does not change much on recovery. Inpari30
can maintain higher chlorophyll levels after submergenceand on recovery. The chlorophyll content of IR72442
decreased during submergence and did not recover well,even after 6 days of re-aeration (Figure 2).
Influence of anaerobic environment on Fv/Fm andphotosynthesis rate
Fv/Fm for the control plants of both varieties did not differ
significantly during the experiment. After 6 days ofsubmergence, Fv/Fm for IR72442 decreased significantly(88.9%), and although the value largely recovered by the endof the 6-day re-aeration period (to 6.11 times thesubmerged value), Fv/Fm remained significantly lower thanthe control. After 6 days submergence, Fv/Fm for Inpari30
decreased marginally (not significant) and then increased
very slightly after 6 days of recovery (Fig. 3A).
After 6 days of submergence, net photosynthesis for bothvarieties decreased significantly versus controls (>41%), andalthough photosynthesis recovered to some extent after 6
days of re-aeration (>27.4%), values remained significantly
lower than controls (Fig. 3A).
Total dry weight changes affected by submergence
Comparison of growth was made via changes in total dry
weight during the submergence period and during therecovery period, expressed as the ratio between submerged(S) and control (C) plants. The S/C ratio for Inpari30 was
higher than that for IR72442 after submergence and after
recovery (Table 1).
The ratio of shoot dry weight per shoot length
The ratio of shoot dry weight and shoot length was the same
between varieties and treatment before exposing tosubmergence. After submergence, submerged IR72442
decreased the ratio significantly lower than control andsubmerged Inpari30, though no significant difference with
control IR72442. After recovery, submerged IR72442 wassignificantly lower than submerged Inpari30, though there isno significant observed with control plant (Fig. 4).

108

The ratio between shoot dry weight and shoot length during
the experiment is explained in Fig 4. IR72442 significantly
increase plant length after submergence highest among all
treatments (Fig. 1). However, the increasing of plant length
was not followed by the accumulation of shoot dry weight.
Thus, the ratio of shoot dry weight per shoot elongation was
lower in submerged IR72442 compared with Inpari30 asshown in Fig. 4.

deterioration of PSII in tolerant cultivars was comparatively
less than susceptible ones. The decline in Fv/Fm for IR72442
during submergence likely reflects a reduced ability of PSII to
reduce the primary acceptor (Panda and Sarkar, 2012). This
provides evidence for disorganization of the photosynthetic
apparatus and is attributed to a decrease in light intensity
and oxygen level in floodwater (Panda et al., 2008; Panda
and Sarkar, 2012). This reduction is also indicative ofphotoinhibition damage in response to environmental stressresulting in a decline in the efficiency of solar energy
conversion during photosynthesis in combination with an
overall decline in photosynthetic capacity (Maxwell &Johnson, 2000; Inamullah and Isoda, 2005). Submergedplants of Inpari30 described to maintain absorbing energy
from light, however plants declined photosynthetic rate bylimiting gas exchange and stomatal closure during anaerobic.
After six days of re-aeration, Fv/Fm for the submerged
IR72442 recovered but only to a level below that of thecontrols. However, the tolerant rice variety, Inpari30, wasable to adjust to the aerobic environment quickly, as
observed from Fv/Fm values (Fig. 2A). During re-aeration,
the photosynthetic rate largely recovered fromsubmergence levels in both varieties, but remained
significantly below those of controls (Fig. 2B). Variablefluorescence (Fv) equals the fluorescence increase fromminimal fluorescence (Fo) to maximal fluorescence (Fm), Fv= Fm – Fo. Then the maximal quantum yield of PS II Fv/Fm isthe function of quantum yield = (Fm – Fo)/Fm (Hall et al.,
1993; Panda et al., 2008). Any changes in Fm or Fo would
consequence with the change of Fv/Fm. However, theincreasing in Fv/Fm of the susceptible variety was not
representative of an increase of PSII efficiency, as a
substitute of minimal fluorescence (Fo) decreasing
compared to maximal fluorescence (Fm) on themeasurement observed (Panda et al., 2008). This describes
the possible cause of increasing Fv/Fm of submerged
IR72442 after six days of recovery.
The photosynthetic rate in both varieties decreased during
submergence; however, Inpari30 was able to groweffectively after submergence as reflected that the dryweight ratio of submerged to control (Table 1). Shoot dry
weight in both varieties declined during flooding treatment,
but after just 6 days of re-aeration, shoot dry weight for
Inpari30 recovered to the same level as the controls. The
higher ratio of shoot dry weight per length on Inpari30
represents that by slow elongation during submergence, thisvariety able to maintain dry matter (Fig 4). This reflects theresults of Sarkar and Bhattacharjee (2011), who found that
Sub1 varieties exhibit the ability to maintain higher biomass
and can re-grow faster during re-aeration.
The narrow alteration of SPAD value and Fv/Fm explainedthat the vast changes photosynthesis does not affect bothvariables at Inpari30, but not at IR72442 (Fig 5A, 5B). This
also means that the response of the SPAD value and Fv/Fm
is more independent than the photosynthesis rate in
explaining the effect of submergence on Inpari30. The vastchange of SPAD and Fv/Fm value in IR72442 as the change ofphotosynthesis rate perhaps related to varietal factors in
response to environmental changes during submergence,such as changes of light intensity that reflected by SPAD and
Fv/Fm measurements. Light intensity changed from 350 to330 μmol m−2 s−1 during submergence which responded by
Inpari30 and IR72442 differently.
Photosynthesis, the transformation of carbon dioxide into
organic matter in the presence of plant chlorophylls under

The changes in photosynthesis rate over SPAD value andFv/Fm
The scatterplot figure represents the relationship between
variables of submerged Inpari30 and IR72442. Given thesignificant changes in the photosynthetic rate observed inboth varieties, the transition in SPAD value and Fv/Fm at
Inpari30 is smaller than IR72442 (Fig 5A, 5B). Plant
physiological response of SPAD value and Fv/Fm tophotosynthetic rate (Pn) was lower in Inpari30 than in
IR72442. Besides, this shows that the response of SPAD
value and Fv/Fm is more independent than thephotosynthesis rate in explaining the effect of submergenceon Inpari30, but not on IR72442.
Discussion
The effect of submergence commonly does not appear
immediately as visual damage, but it soon becomes
observable after the water level recedes (Drew, 1997). The
first noticeable effect of submergence was the increased
plant length of IR72442, greater than Inpari30 in thesubmergence compared to control (Fig. 1). According to themechanism of submergence defined by Nagai et al. (2010)
and Hattori et al. (2011), the elongation exhibited by
IR72442 represents a transient submergence intolerance
whereas the relative quiescence exhibited by Inpari30
represents tolerance. Inpari30 carries the gene Sub1A(Septiningsih et al., 2015), which limits plant elongation
during submergence, enabling a greater accumulation ofshoot dry matter than IR72442 on recovery, as indicated by
the S/C ratio (Table 1), and it allows the plant to conserve
energy for regrowth once the water level subsides. This
result contrasts with that of Sarkar et al. (1996) who
reported that submergence-tolerant cultivars accumulated
more carbohydrates before submergence to elongate theseedling height allowing the production of new
carbohydrates as the leaves became exposed to sunlight
above the water. In varieties exhibiting shoot elongation, thedry weight of leaves developed before submergence is used
for leaf elongation (Kawano et al., 2008). In this study,
IR72442 showed extensive shoot elongation, with low ratioof dry weight per shoot elongation (Fig 4), despite a decline
in photosynthetic rate observed during submergence (Fig.
2B).
Chlorophyll fluorescence (Fv/Fm) is an effective indicator ofsubmergence tolerance of rice (Sone et al., 2012). In thisstudy, Fv/Fm for the controls (i.e. non-stress conditions) did
not differ significantly between Inpari30 and IR72442 (Fig.
2A); therefore, the decrease in Fv/Fm of IR72442 during
submergence can be attributed to the submergencetreatment. Conversely, net photosynthesis was rapidly
decreasing during submergence for both Inpari30 and
IR72442 versus controls (Fig. 2B). Significantly decline of Fvvalue in the susceptible cultivar than tolerant cultivar also
reported by Panda et al. (2007) which then assumed that109

Table 1. Ratio of total dry weight after submergence and after recovery (re-aeration).
Treatment

Total dry weight aftersubmergence (g plant−1)

C Inpari30
S Inpari30
C IR72442
S IR72442

0.01880.01440.01800.0105

S/C ratio
0.76 a0.59 b

Total dry weight after
recovery(g plant−1)
0.0152 d
0.0326 a
0.0242 bc
0.0259 b

S/C ratio
2.15 a1.07 bNote: C, Control; S, Submerged. Total dry weight after submergence is the difference value between total dry weight after submergence and before
submergence. Total dry weight after recovery is the difference value between total dry weight after recovery and after submergenceFig 1. Plant length of two contrasting rice varieties (IR72442, an elongation type; Inpari30, carrying gene Sub1A) before
submergence, after submergence, and after recovery (re-aeration). Different lowercase letters in the same column indicate asignificant difference at p < 0.05 with Tukey test. Error bar represent standard deviation from three replications. C, Control; S,
Submerged.

Fig 2. Chlorophyll content (SPAD Value) of two contrasting rice varieties (IR72442, an elongation type; Inpari30, carrying gene
Sub1A) before submergence, after submergence, and after recovery (re-aeration). Different lowercase letters in the same column
indicate a significant difference at p < 0.05 with Tukey test. Error bar represent standard deviation from three replications. C,
Control; S, Submerged.

Fig 3. Fv/Fm (A) and net photosynthesis rate (B) of two contrasting rice varieties (IR72442, an elongation type; Inpari30, carrying
gene Sub1A) before submergence, after submergence, and after recovery (re-aeration). * and ** in each the stage of observation
indicates significance of differences between control and submergence in p < 0.05 and p < 0.01, respectively. Different lowercase
letters in the same column indicate a significant difference at p < 0.05 with Tukey test. Error bar represent standard deviation fromthree replications. C, Control; S, Submerged.

110

Fig 4. Accumulation of shoot dry weight per shoot length of two contrasting rice varieties (IR72442, an elongation type; Inpari30,
carrying gene Sub1A) before submergence, after submergence, and after recovery (re-aeration). Different lowercase letters indicate
a significant difference at p < 0.05 with Tukey test. Error bar represent standard deviation from three replications. C, Control; S,
Submerged.

Fig 5. Relationship between net photosynthesis and (A) SPAD value, and (B) Fv/Fm of submerged Inpari30 and IR72442 rice plants.
Triangle shape represent submerged IR72442 and circle represent submerged Inpari30.

the influence of light, involves the release of oxygen fromwater (Gest, 2002). Chlorophyll converts the energy from
captured light photons into chemical energy that is used todrive CO2 fixation (Murchie and Lawson, 2013). Under
laboratory conditions, the transport of PSII electron and the
fixation of CO2 can be very well associated. However, thisconnection may result in difference under field condition.
Differences can be caused by changes in the relative levels of
CO2 fixation and by competing processes such as
photorespiration, nitrogen metabolism and oxygen donation(Maxwell and Johnson, 2000). The electrons released during
the photochemical process are not always used for carbon
fixation when environmental factors perturb CO2 availability(e.g. under anaerobic conditions, stomatal closure, etc.). In
these circumstances, the excitation of electrons could beconsidered as a waste of energy, potentially producing
reactive oxygen species leading to oxidative stress. The
disturbance of electron transfer can be recognized fromdecreasing quantum yield of electron transfer in PSII
(Fv/Fm), this was observed during submergence in IR72442
but not in Inpari30 despite the photosynthetic rate being in
decline in both (Fig. 2A; 2B) and the relation both variables
explained (Fig 5B). This means that even though Fv/Fm could
identify PSII efficiency in a tolerant variety, it could not be
used to predict an accurate determination of CO2 fixation
with fluorescence alone is not possible (Maxwell andJohnson, 2000).

temperature during the experiment was maintained 27C
with 350 μmol m−2 s−1 of 12 h light exposure.
Experimental design
A randomized complete block design was employed in thisexperiment. Two factors were studied. The first factor wasenvironmental conditions: control versus submergence. The
second factor was rice variety, which was fully randomizedon the environmental conditions. To compare different
tolerance mechanisms during complete submergence
followed by several days of re-aeration, we used rice variety
Inpari30 as a Sub1A genotype and variety IR72442 as anelongation type. The experiment was conducted in three
replications.
Experimental setup and conduction
Rice seeds of Inpari30 and IR72442 were soaked andincubated for three days at 30C. Germinated seeds werethen sown into commercial soil for rice seedling (N:P:K =0.9:2.3:1.1; pH 4.5–5.2) in a growth chamber. Ten-day-old
seedlings were transplanted into a sponge and inserted intoa seedling tray inside experimental glass containers. The
water level was maintained at 4.5 cm from the container
base, the same level as the seedling tray surface also theplant stem base. The experiment was conducted in acontrolled room with 12 h light exposure at 350 μmol m−2 s−1
measured 20 cm above the tray surface and at 27C
(Day/Night). However, under submergence the light
exposure was approximately 200 μmol m−2 s−1 lower. The
oxygen concentration at the same water depth was ranging
from 7.0-8.9 mg L-1 during the experiment.
At 14 days after seeding (DAS), when the third leaf was fully
expanded, the water level in the glass container was raised

Material and methods
Location
The research was conducted from June to July 2018, in
controlled room conditions at the Tropical Crop Science
Laboratory, Kagoshima University, Japan. The daily111

to 35 cm above the shoot base for the plants receiving
treatment, while the water level for the control plants wasmaintained as height as the stem base. Treatment was
stopped after 6 days (20 DAS), at which point the water level
for treatment and control was maintained at the same level,
adjusted to the stem base. The recovery stage lasted 6 days
which after that all the plants were removed from theircontainers (i.e. at 26 DAS). Seedlings were only exposed bysubmergence only for 6 days allowing measurement thewhole process of stress effect and recovery without plant
death as the worst effect of continuous submergence.
Measurement of Fv/Fm, SPAD value and photosynthesis rate
were performed on the same leaf, the 3rd leaf, as the fully
expanded leaf before treatment started, those thedeveloping of 3rd leaf during submergence and recovery
could be used to evaluate the effect of stress exposure.
Tillers were not developed yet during the whole experiment
therefore data were not available.

Gas exchange measurements
Measurement of net photosynthesis (PN) was assessed using
a portable photosynthesis analysis system (LI-6400; LI-COR,
Lincoln, NE, USA). Before measurements were made, plant
samples were adapted under light exposure of 300-350μmol m−2 s−1 same as growth environment condition. The
third leaf of each sample was selected and kept inside thechamber until a stable reading was recorded. Leaf area wasmeasured to calculate the gas exchanges rate per observed
area. During measurement, relative humidity was ~50%, leaf
temperature 25C, ambient CO2 concentration ~400 μmol
mol−1, airflow through the chamber was maintained at 500
mol s−1, and photosynthetic photon flux density (PPFD)
1100 μmol m−2 s−1.
Total dry weight (g)Four plant samples from each replicate were acquired for
total dry weight measurement. The plant samples were put
in envelopes and dried in an oven for 72 h at 80C. The ratio
S/C (submerged/control) for weight change was thencalculated for the period of submergence and the period ofrecovery.

Variables analysed
PS II quantum yield (Fv/Fm)
After 2 hours of dark adaptation, equipment to measurechlorophyll fluorescence (AquaPen-P AP-P 100, PSI, Czech
Republic) was attached to the third leaf of the rice plant
being sampled. The maximal quantum yield of PS II
photochemistry (Fv/Fm) was obtained by actinic lightemission through the quantum yield (QY) menu. Variablefluorescence (Fv) equals the fluorescence increases fromminimal fluorescence (Fo) to maximal fluorescence (Fm), Fv= Fm – Fo. Then the maximal QY of PS II Fv/Fm is thefunction of QY = (Fm – Fo)/Fm (Hall et al., 1993; Panda et al.,2008). Any changes in Fm or Fo would consequence with thechange of Fv/Fm. The maximal fluorescence (Fv/Fm) value is0.85 that equals 85% efficiency of the conversion ofabsorbed light into photochemistry.

Statistical analysis
Data were analysed from three replications using two waysof analysis of variance. If significant differences were found,the Tukey test was performed at p = 0.05.
Conclusion
Submergence decreased net photosynthesis both Inpari30
and IR72442. However, Inpari30 maintain stable shootelongation as well as Fv/Fm and SPAD value during
submergence. Plant physiological changes of SPAD value andFv/Fm to photosynthetic rate (Pn) was lower in Inpari30
than in IR72442. Moreover, Inpari30 accumulated higher dry
weight per shoot elongation. In conclusion, the Sub1
genotype of Inpari30 confers the ability to maintain Fv/Fm
under complete submergence and in conditions limiting gas
exchange for photosynthesis.

Leaf chlorophyll content (SPAD value)
The amount of chlorophyll present in leaves was estimatedusing a leaf chlorophyll meter (SPAD-502, Konica Minolta
Corporation, Japan). The chlorophyll content wasrepresented by the average Soil Plant Analysis Development(SPAD) value of three measurements on the tip, middle, andbase of the third leaf. The strong relationship between SPAD
index and leaf chlorophyll concentration has been widelyreported on several. This tool measures the chlorophyll
content of the leave without destruction, simple, quick, andportable. Moreover, a significant linear regression line wasreported between SPAD reading and chlorophyll content inrice (Kumagai et al., 2009). The leaf chlorophyll content isaffected by N status in the plant that commonly has a strong
positive relationship with the photosynthetic rate (Sinclair
and Horie, 1989). Therefore, SPAD reading is possible to use
as an indirect countermeasure of photosynthetic capacity on
the rice leaf.

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