Ghorbanzadeh 

et al. BMC Genomics (2023) 24:152 BMC Genomics

https://doi.org/10.1186/s12864-023-09246-z

RESEARCH Open Access

Comparative metabolomics of root‑tips

reveals distinct metabolic pathways conferring
drought tolerance in contrasting genotypes
of rice
Zahra Ghorbanzadeh1, Rasmieh Hamid2, Feba Jacob3, Mehrshad Zeinalabedini1,
Ghasem Hosseini Salekdeh1,4 and Mohammad Reza Ghaffari1* 

Abstract 
Background  The mechanisms underlying rice root responses to drought during the early developmental stages are
yet unknown.
Results  This study aimed to determine metabolic differences in IR64, a shallow-rooting, drought-susceptible geno-
type, and Azucena, a drought-tolerant and deep-rooting genotype under drought stress. The morphological evalu-
ation revealed that Azucena might evade water stress by increasing the lateral root system growth, the root surface
area, and length to access water. At the same time, IR64 may rely mainly on cell wall thickening to tolerate stress.
Furthermore, significant differences were observed in 49 metabolites in IR64 and 80 metabolites in Azucena, for
which most metabolites were implicated in secondary metabolism, amino acid metabolism, nucleotide acid metabo-
lism and sugar and sugar alcohol metabolism. Among these metabolites, a significant positive correlation was found
between allantoin, galactaric acid, gluconic acid, glucose, and drought tolerance. These metabolites may serve as
markers of drought tolerance in genotype screening programs. Based on corresponding biological pathways analysis
of the differentially abundant metabolites (DAMs), biosynthesis of alkaloid-derivatives of the shikimate pathway, fatty
acid biosynthesis, purine metabolism, TCA cycle and amino acid biosynthesis were the most statistically enriched bio-
logical pathway in Azucena in drought response. However, in IR64, the differentially abundant metabolites of starch
and sucrose metabolism were the most statistically enriched biological pathways.
Conclusion  Metabolic marker candidates for drought tolerance were identified in both genotypes. Thus, these mark-
ers that were experimentally determined in distinct metabolic pathways can be used for the development or selec-
tion of drought-tolerant rice genotypes.
Keywords  Root Tips; Rice, Drought Stress; Root Architecture, Metabolites

*Correspondence:
Mohammad Reza Ghaffari
mrghaffari52@gmail.com
Full list of author information is available at the end of the article

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Ghorbanzadeh et al. BMC Genomics (2023) 24:152
Introduction
Drought is a major abiotic stress that restricts crop
growth, development and yield and thus has turned into
a grave threat to universal food security [1]. In addition,
global climate change, particularly high temperatures
and erratic rainfall patterns, combined with a growing
world population, is placing tremendous stress on food
security and sustainability. These challenging conditions
can be overcome through breeding programs to develop
drought-resistant crops [2, 3]. The combined effect of
drought and other abiotic stresses can reduce potential
crop production by more than 50%. According to mod-
eling simulations, drought-affected cropping regions
could quadruple by the end of the twenty-first century
[4]. In response to these conditions, rice employs various
adaptive methods, such as building up various osmopro-
tectants or solutes and changes in the direction of plant
growth to avoid drought [5]. Roots are the plant’s main
organs that anchor it in the soil and are required for
nutrition and water absorption. Favorable responses of
plants to water stress is dependent on the roots’ capac-
ity to maintain growth (i.e., modifying the root traits
such as depth, density, and root angles) and maintain/
increase root hydraulic conductivity [6–8]. The ability of
roots to tolerate water deficiency depends on their abil-
ity to maintain adequate carbohydrate metabolism, cell
wall protein composition, osmotic potential, and metab-
olites involved in the oxidative stress response [9, 10].
Most research has been focused on improving features
in above-ground tissues to tolerate these pressures, but
roots (the ‘hidden half ’ of a plant’s architecture) remain
an underutilized source of crop development [11]. Root
System Architecture (RSA) is critical for improving
nutrient and water uptake and maintaining crop yield
under optimal and drought conditions [12]. Extensive
root systems can help plants overcome drought, which
is influenced by growth angle, root thickness, and length
[13, 14]. The meristematic zone of root tips is ideal for
studying these root traits because of its high mitotic rate
[15]. Lowland rice with a shallow rooting system is more
susceptible to drought stress, whereas upland rice with
a deep rooting system is more drought-tolerant [16].
Understanding the adaptive reactions of different root
sections is critical for generating better stress-tolerant
rice. The omics technologies, viz. genomics, transcrip-
tomics, proteomics, and metabolomics, have been used
to uncover metabolic pathways that could be altered to
overcome the effects of drought stress [16–18]. Metab-
olomics is a potent tool for obtaining extensive data
on metabolite profiles and metabolic network analysis
[19]. Various metabolite profiling methods are used to
understand the plant molecular responses to drought
stress and determine the metabolites’ levels in a specific
Page 2 of 20
metabolite pathway or class [20–22]. The metabolic pro-
file of drought-stressed barley and maize leaves and roots
indicated a substantial buildup of metabolites belong-
ing to glyoxylate and dicarboxylate metabolism in maize
roots and isoflavonoid biosynthesis in barley roots [23–
25]. Further findings revealed that drought stress toler-
ance is linked to a signature of metabolites of different
metabolic pathways [20]. Plant drought tolerance has
been thoroughly characterized at various levels, but the
metabolite-mediated regulatory mechanisms involved
in the RSA traits of upland rice remain a mystery. Root
tips have a developmental gradient from meristematic to
mature cell zones, each of which responds to water stress
differently and serves a specific purpose in the formation
of RSA [26, 27]. Among the different zones, the meris-
tematic zone is the most critical zone for determining
roots’ length, angle, and thickness [28]. Thus, investigat-
ing the changes in the metabolite profile of the meris-
tematic zone could be a practical approach to identifying
metabolites related to drought tolerance and could help
develop drought-tolerant varieties [29]. For the current
study, a metabolomics approach was employed to deter-
mine the alteration of the corresponding metabolites in
the root tip (5 mm) regions of IR64, a stress susceptible,
shallow-rooted lowland genotype, and Azucena, a stress-
tolerant, traditional upland genotype, grown in con-
trol and drought conditions to get additional molecular
insights on how drought affects metabolite production.
Root-tip differentially abundant metabolites (DAMs)
suggested a distinct pattern of metabolite accumulation
which confers drought tolerance to rice.
Results
Root morphological changes induced by drought stress
To evaluate the changes in root morphology and meta-
bolic content of the two genotypes, samples were
treated with severe drought stress (30% FC). Table  1
shows that the phenotypic responses of roots of both
genotypes to drought stress were significant statistically
(p-value < 0.05). Drought stress increased the mean root
length and root surface area, lateral root development,
and primary and secondary root diameters in Azu-
cena (Table  1). While slightly relaxed P-value of 0.05 to
no statistically detectable lateral root formation, diam-
eters of the primary and secondary roots were observed
in IR64. The root length in Azucena grew from 30.1 cm
to 49.18 cm after drought stress induction, whereas this
change was prolonged in IR64 (25.14  cm to 29.09  cm)
(Table  1). Similar results were observed for the surface
area parameter; thus, the mean surface area increased
from 90.77 c­m2 to 115.87 c­m2 in stressed Azucena,
whereas it changed from 70.01 c­ m2 to 77.61 c­ m2 in IR64.
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 3 of 20

Table 1  Phenotypic parameters of the roots assessed for both genotypes using the SmartRoot system
Genotype Treatment ARA​ ARW​ ARH RL SA N.Tip LR% RD t-test

IR64 Control 41.08 ± 1.01d 43.09 ± 2.08d 60.70 ± 2.47d 25.14 ± 1.19c 70.01 ± 2.14d 27 ± 1.17d 35.09 ± 2.98d 0.30 ± 0.02c 0.017
c b b b c c b,c
Drought 47.10 ± 1.12 53.01 ± 1.18 75.25 ± 1.61 29.09 ± 1.04 77.61 ± 1.10 30 ± 0.09 46.12 ± 3.13 0.35 ± 0.01b 0.048
b c c b b b b
Azucena Control 52.14 ± 1.26 46.35 ± 1.49 70.15 ± 2.29 30.01 ± 2.87 95.77 ± 3.07 35 ± 2.11 50.87 ± 4.75 0.35 ± 0.04b 0.014
a a a a a a a
Drought 59.49 ± 1.50 56.10 ± 1.15 90.25 ± 4.85 49.18 ± 3.14 115.87 ± 3.02 48 ± 3.76 89.14 ± 3.69 0.46 ± 0.02a 0.010
ARA​Analyzed Region Area ­(cm2), ARW​ Analyzed Region Width (cm), ARH Analyzed Region Height (cm), RL Root Length (cm), SA Surface Area (­ cm2), N. Tip number of
tips, LR lateral root (%), RD root diameter (cm). In each column, the same letters do not differ at significance level P < 0.05 (Duncan’s multiple range test)

Table 2 Explanation and predictability values of the principal derivatives, benzoxazinoids, fatty acid, flavonoid, lipids,
component analysis (PCA) and partial least squares-discriminate nucleic acid derivatives, organic acid, phenolamides,
analysis (PLS-DA) polyphenols, sugar, sugar alcohol, vitamins and non-
Control- Control- Control- Stress (Azu & IR) classified (Table S1). The metabolic data of the AZs, AZc,
stress (Azu stress stress IRs, and IRc groups were subjected to PCA, an unsuper-
& IR) (Azu) (IR) vised multivariate data analysis method to decrease the
PCA R2X 0.713 0.742 0.759 0.659 data’s dimensionality and visualize the sample grouping.
Q2 0.497 0.4015 0.462 0.4055 Four principal components (PCs) with explanatory and
PLS-DA R2X 0.586 0.485 0.672 0.501 predictive values of 71.3% and 49.7% each were used to
R2Y 0.512 0.901 0.906 0.588 build the PCA model (Table 2). The score plot of the first
Q2 0.471 0.717 0.794 0.086 two PCs is shown in Fig. 1A. The majority of the data fell
inside the 92% confidence interval (Hotelling T2 ellipse).
The PCA results for the four cluster samples showed that
there was a clear distinction between the control (c) and
The numbers of root tips were also more pronounced in
treated samples (drought-stressed), while no clear differ-
Azucena than in IR64 (Table 1).
ence was seen between the two genotypes (Fig.  1A). To
confirm this trend, three of the whole competitive groups
Metabolic changes in roots in response to drought stress were examined by PCA, yielding similar results. The
Metabolites of root tip (5 mm) regions were subjected to PCA models yielded two, two, and three PCs, respec-
GC–MS to measure a wide range of metabolic changes tively, for comparing AZs vs. AZc, IRs vs. IRc, and AZs
of drought-stressed Azucena (AZs), control Azucena vs. IRs samples, respectively. The ­R2 X and Q­ 2 (goodness
(AZc), drought-stressed IR64 (IRs), and control IR64 of prediction) values are shown in Table  2 and indicate
(IRc). In total, 156 metabolites were identified unambigu- that the differences between groups could be predictably
ously and included, alkaloids, amino acids, amino acid explained by all the models. However, no distinct limit

Fig. 1  Principal component analysis (PCA) and Partial least squares-discriminate analysis (PLS-DA) score plots of metabolic profiles in rice roots
under drought stress. A PCA score plot for Azucena normal (red), IR64 normal (blue), Azucena-treated (yellow) and IR64-treated (green) samples, B
PLS-DA score plot for Azucena normal (red), IR64 normal (blue), Azucena-treated (yellow) and IR64-treated (green) samples
Ghorbanzadeh et al. BMC Genomics (2023) 24:152
could be seen between the two groups’ PCA score plots
(Table  2). PLS-DA is a supervised method that catego-
rizes the observations into groups that yield the largest
predicted indicator variable. The obtained data resulted
in two PCs (­R2 X  =  0.586, ­R2 Y  =  0.512, ­Q2 = 0.471)
between the four cluster, and enhanced the classifica-
tion between these groups in the score plot (Fig.  1B). It
led to better modeling and prediction results with two
PCs, when the data were examined with only the control
and the treated-AZ or treated-IR64 ­(R2 Y > 0.9, ­Q2 > 0.7)
(Table 2) As shown in Table 2, the treated AZ and IR sam-
ples could be separated with two PCs in spite of the slight
overlap in the PLS-DA score plot (R2 Y > 0.5, Q2 > 0.08),
indicating intrinsic metabolic differences between these
two genotypes in the treated conditions.
Overview of Azucena and IR64 root‑tips metabolome
under control and drought stress conditions
The line plots of the X-loadings of the first component of the
PLS-DA pairwise comparison models were used to identify
the primary altered metabolites. Based on the parameter
VIP > 1, p-value ≤ 0.01, log fold change (log FC > 2.0), and
Kruskal–Wallis ANOVA, a total of 103 drought respon-
sive metabolites with significant differences were identified
(Table S2 and S3). The most significant metabolites had
variable importance in the projection (VIP) values greater
than 1, which was reported to explain the responses [30].
The VIP values for metabolites categorized by superclass are
shown in Fig. 2A. Amino acid and organic acid groups were
most abundant. For example, GABA and aspartic acid had a
VIP value of 20.52 and 18.30, although the average value of
the exclusive VIP was 5.63. Organic acid (16%), amino acid
Fig. 2  Classification of the differentially abundant metabolites and their variable importance in the projection (VIP) distribution in IR64 and
Azucena in root tips. A VIP distribution in each metabolite superclass as a scatter plot. The average mean of the differentially abundant metabolites
is shown in the red dashed line (B) A pie chart depicting the proportion of each metabolite in the superclass
Page 4 of 20
(12.2%), polyphenols (12.8%), nucleic acid derivatives (8.3%)
and unclassified (others, 7.1%) were the most common dif-
ferentially abundant metabolites (Fig. 2B).
Compared to the control condition, the levels of 56 metab-
olites increased and 24 metabolites decreased in the Azucena
root-tips in response to drought stress. In contrast, in IR64,
the levels of 30 metabolites increased, and 19 decreased in
response to drought stress (Fig. 3A). A cross-comparison of
the differentially abundant metabolites between genotypes
showed that number of accumulated metabolites were nearly
two times higher in the root tips of the tolerant genotype (53
metabolites) than the sensitive one (22 metabolites), and of
which 27 metabolites were commonly altered between the
two genotypes in response to drought stress (Fig.  3B). The
25 most differentially abundant metabolites are shown in
Fig. 4. The ­log2 fold change values and VIP score for these
metabolites are mentioned in Table S2 & Table S3. As shown
in the Fig. 4, the levels of amino acids including aspartic acid
and glutamic acid, nucleotides namely thymine and guanine
increased in both genotypes under drought stress. Inter-
estingly, glycine, phenylalanine, threonine, isoleucine, and
GABA were accumulated in IR64, while there was no sig-
nificant difference in the levels of these metabolites in Azu-
cena under drought stress. Likewise, there is a considerable
increase in the levels of TCA cycle intermediates comprising
of malic acid, isocitric acid, succinic acid and fumaric acid,
and sugar and sugar alcohol such as sorbitol, mannitol, galac-
tinol, myo-inositol, D-raffinose, sucrose, ribose and treha-
lose. In particular, the levels of several metabolites involved
in secondary metabolism comprising of genistein, vanil-
lin, scopoletin, conifery aldehyde, farnesyl pyrophosphate,
betaine, cyanidin 3-O-rutinoside 5-O-beta-D-glucoside, and
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152
Fig. 3  Overview of differentially abundant metabolites (DAMs) between the Azucena and IR64. A Up- and down-regulated metabolites of Azucena
and IR64 in response to drought stress. B Venn diagram showing the overlap between DAMs responsive to drought stress
syringic acid showed the highest levels in the drought toler-
ant cultivar Azucena.
Identification of potential association between metabolites
and observed root traits
Metabolite content was determined in the root tips of
control and drought-stressed plants from two different
genotypes. The hierarchical clustering for both metabo-
lites and samples (genotypes × conditions) is shown in
Fig. 5. Clustering of the samples showed complete sepa-
ration of the metabolite pattern between the control
and drought-treated samples. Thus, drought treatment
was the main source of variance in the data, indicating a
complete change in metabolism under stress conditions
in both genotypes. Figure  5 shows the metabolites that
changed significantly under drought stress; glutamic acid,
aspartic acid, proline, glucose-6-phosphate, and thymine
were among the predominant metabolites that increased
in response to drought stress, whereas metabolites
belonging to quinic acid and ribonic acid (lowest group
in Fig. 5) decreased under drought stress.
The potential correlation between the abundant
metabolites and the root phenotype were tested by ana-
lyzing the correlations of expression of the metabolite
levels with phenotypic traits. We rely on the predic-
tion marker for high concentrations since high amounts
of a metabolite can be detected more accurately than
low concentrations or their absence. High metabo-
lite concentrations in tolerant cultivars are indicated
by significant positive correlations of metabolites with
phenotypic traits, whereas high concentrations in sus-
ceptible cultivars are indicated by significant negative
correlations. In a positive correlation, the metabolite
Page 5 of 20
would be a tolerance marker because its higher concen-
trations would contribute to the tolerance. Metabolites
with negative correlation are sensitivity markers. Most
of the significant correlations between metabolite lev-
els and root phenotypic parameters were found to be
positive under drought stress (Fig.  6). Positive correla-
tions were observed for the concentration of the treha-
lose, proline, succinic acid, tryptophan, salicylic acid,
sucrose, fructose lysine and mannitol. Higher concen-
trations of these metabolites were associated with the
number of tips, root length (cm), surface area ­(cm2),
and root diameter. In tolerant plants as opposed to sen-
sitive plants, concentrations of these metabolites were
higher during drought stress.
However, for most of these metabolites, no correla-
tions were found between concentrations under control
conditions and root performance under drought. In
contrast, concentrations of ribose under control con-
ditions correlated positively with performance under
drought and since there was a positive correlation
between concentrations and root traits performance
under control conditions also, ribose concentrations
appear to be related to rate of root growth rather than
drought tolerance. Gallic acid and ascorbic acid made
better candidates for drought markers because their
concentrations exhibited positive correlation with
root performance only under drought stress condi-
tions. Negative correlations were found for concen-
trations of leucine, isoleucine, pyroglutamic acid,
phenylalanine, and glycerol. Higher concentrations of
these metabolites were associated with decreased root
length, surface area, and number of root tips. However,
GABA concentration was strongly correlated with root
Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 6 of 20

Fig. 4  A Bar graph of 25 differentially abundant metabolites in Azucena root tips. VIP values are in a blue column and the red columns represent
log2 (fold change, FC) values. B Bar graph of 25 differentially abundant metabolites in IR64 root tips. VIP values are in a blue column and the red
columns represent log2 (fold change, FC) values
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 7 of 20

Fig. 5  Metabolite response to drought differs between the two rice cultivars. Hierarchical clustering and heatmap of metabolite levels in root tip of
IRc: IR64 genotype under control condition, IRs: IR64 genotype under drought stress conditions, AZc: Azucena genotype under control conditions,
AZs: Azucena genotype under drought stress conditions
Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 8 of 20

Fig. 6  Correlation of root phenotypic data with metabolite levels. Correlation coefficients for selected metabolites with significant (p≤0.05) positive
(blue) or negative (red) correlation between concentration metabolite levels with root phenotypic data under drought or control conditions. Data
of root tip of two cultivars (Azucena and IR64) grown in two conditions. Mean values of three to five replicates per cultivar and condition were
correlated. ARA (Analyzed Region Area (cm2)); ARW (Analyzed Region Width (cm)); ARH (Analyzed Region Height (cm)); RL (Root Length (cm)); SA
(Surface Area (cm2)); N.Tip ( number of tips), LR ( lateral root (%)), RD (root diameter (cm)

diameter. The levels were 10 to 100 times higher in the
sensitive genotype than in the tolerant genotype.
Functional annotation of DAMs
The differentially altered metabolites were functionally cat-
egorized based on the DAMs in Azucena and IR64, respec-
tively, according to the KEGG database (www.​kegg.​jp/​kegg/​
kegg1.​html). When the control and treated plants were
compared, it was observed that drought stress significantly
altered the relative abundance of the levels of several metab-
olite classes. The most represented categories were organic
acid compounds, amino acids, polyphenols, flavonoids, and
sugars. Between the Azucena and IR64 genotypes, there was
a significant variation in the proportion of organic acid com-
pounds and biosynthesis of carbohydrates and amino acids.
The metabolites in Azucena were mainly associated with
amino acid biosynthesis and the TCA cycle, aminoacyl-
tRNA biosynthesis, and fatty acid biosynthesis (Fig. 7) while
metabolites in the IR64 genotype were mainly related to
phenylalanine and galactose metabolism (p < 0.05) (Fig.  8).
Increase in sucrose, glucose, tryptophan, and proline in
the metabolic pathways of Azucena, may have a substantial
impact on how resistant Azucena is to drought. Drought
stress can have severe consequences for phenylalanine pro-
duction in IR64. The high GABA expression together with
its negative string correlation with root traits also indicates
that IR64 restricts root elongation. Candidate DAMs dem-
onstrating important functions or variant-specific expres-
sion profiles in Azucena and IR64 are listed in Table S2 and
S3, and their relationships to major functional categories are
shown in Fig. 9. A total of 103 DAMs were enriched in 30
metabolic pathways, which were most strongly represented
by starch and sucrose metabolism, amino acid biosynthesis,
secondary metabolite biosynthesis, purine metabolism, and
fatty acid biosynthesis.
Expression of genes involved in drought stress response
Potential marker genes involved in sugar metabolism,
root growth and elongation were identified in our pre-
vious transcriptome studies on two rice genotypes with
different drought tolerance [31, 32]. Thirteen genes were
selected as candidate markers, and their expression
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 9 of 20

Fig. 7  KEGG pathway classification of the differentially abundant metabolites under drought stress in Azucena (www.​kegg.​jp/​kegg/​kegg1.​html)

Fig. 8  KEGG pathway classification of the differentially abundant metabolites under drought stress in IR64 (www.​kegg.​jp/​kegg/​kegg1.​html)

levels were measured by qRT-PCR in the roots of the two
genotypes grown under control and drought stress con-
ditions. It was found that the gene Ethylene response fac-
tor (ERF35) was significantly expressed in the Azucena
genotype (5.63-fold), whereas it was less expressed in the
IR64 genotype (3.56-fold). Gene expression of serine/
threonine kinase (SnRK2), IAA19, Trehalose -phosphate
phosphate1 (OsTPP1), SUCROSE TRANSPORTER 5
(SUT5), dehydration responsive element binding protein
(DREB) increased (upregulated) in Azucena by 4.86-fold,
5.38-fold, 5.90-fold, 8.45-fold and 6.75-fold, respectively,
while they had no detectable change in IR64 (Fig.  10).
Primer sequences and more details about genes can
be found in Table S6. To find the interrelation between
expression levels of selected genes and metabolites, the
correlation analysis was done. The connections between
Ghorbanzadeh et al. BMC Genomics (2023) 24:152 Page 10 of 20

Fig. 9  Primary metabolism responses in two rice genotypes (Azucena and IR64) after drought stress. Colors depict the relative accumulation levels
of each metabolite: white (no significance), red (increase) and blue (decrease)

Fig. 10  Validations of selected genes using qRT-PCR in root tip zone of both genotypes, Azucena and IR64, in response to water stress
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152
metabolite levels and expression levels were in the posi-
tive rather than in the negative direction indicating a
co-regulation at both the transcriptional and metabolite
levels. For instance, there was a significant positive cor-
relation between the level of proline and expression of
the pyrroline-5-carboxylate reductase (P5CR) which in
involved in proline metabolism (Fig.  11). Further posi-
tive connections were found between PRP5, glycine- and
proline-rich protein 3 (OsGPRP3), trehalose phosphate
phosphate1 (TPP1), sucrose transport protein (SUT5),
and auxin-responsive protein IAA19 (IAA19) with treha-
lose, sucrose, and tryptophan, which were present at high
levels under drought condition (Fig.  11). Thus, the high
expression of these genes indicates drought tolerance
under drought stress conditions.
Discussion
Contrasting drought tolerances of the two rice genotypes
The effectiveness of water uptake from a diverse soil envi-
ronment is determined by root architecture. In drought
condition due to increased soil resistance and decreased
water availability, roots are unable to absorb or release
water to the soil when the soil dries out. This leads to a
Fig. 11  Correlation of the expression level of selected genes with the abundance of metabolites. Correlation coefficients for selected metabolites
with significant (p ≤ 0.05) positive (blue) or negative (red) correlation between metabolite concentration and expression level of selected gene data
under drought conditions
Page 11 of 20
decrease in osmotic potential and matric potential [33],
as well as a decrease in turgor pressure and cell volume
[34]. Root cells need to develop techniques to counter-
act water loss and its consequences. At times, the solu-
tion potential of the cells is reduced, increasing turgor
pressure and allowing development to continue under
water-deficient conditions [14, 35, 36]. The tiered struc-
ture of the root system can increase hydraulic lift and
thus enhance the water uptake from deeper soil layers
[37]. With the developments in breeding drought-toler-
ant plants, it is beneficial to focus on the characteristics
of the root and patterns of root growth at different loca-
tions and time [38]. The morphological and metabolic
responses of two rice genotypes with different drought
stress responses were examined in this study under con-
trol and drought stress conditions. Total root growth,
average root diameter, root length, and surface area were
the primary morphological traits studied. Improved ver-
tical root formation under water stress has been shown
to benefit crop production [39]. Irrigation regimes have a
significant effect on bread wheat root density and depth
via soil water content [40]. In addition, cotton under
drought stress formed thinner fine roots but longer root
Ghorbanzadeh et al. BMC Genomics (2023) 24:152
hairs [41]. According to these studies, under drought
stress conditions, Azucena has longer roots and a large
surface area for water uptake and storage. The average
root length of this genotype varies from 35 to 50  cm,
while the root diameter ranges from 35 to 46 μm. These
values are almost double than those of IR64. These dif-
ferences indicate that the two genotypes have different
drought resistance mechanisms. Although root length
and surface area have an effect on soil resource uptake
[42], the most important sites for water uptake are the
immature root tips [43]. The uninterrupted growth of
root tips may be necessary for water and nutrient uptake
[44]. In this study, different metabolites were detected
in the two genotypes under drought stress. On the basis
of the PLSDA analysis, a clear distinction was found
between the metabolite content of different rice geno-
types under two different water conditions. In addition to
the metabolites that showed similar patterns of increase
or decrease in both the rice genotypes, a few metabo-
lites showed differences among the two genotypes. In
response to water stress, 80 and 49 metabolites were
detected in Azucena and IR64, respectively. In Azucena,
56 specific metabolites were accumulated in root tips,
whereas in IR64, 30 different metabolites were accu-
mulated. These results suggest that different metabolite
mechanisms are involved in rice genotypes of contrast-
ing drought tolerance. These findings suggest that rice
genotypes with varying drought tolerances are affected
by various metabolic pathways.
DAMs involved in the biosynthesis of carbohydrates
and energy metabolites
Increased levels of energy and carbohydrate metabo-
lites, including those involved in sugar metabolism,
were found in the Azucena in this study. Sugar is essen-
tial for plant development and affects every stage of the
life cycle of the plant. It controls growth and develop-
ment of stressed plants, by interacting with the plant
hormones [45]. Lower sugar accumulation can lead to
weak root system in IR64 genotype, while higher con-
centration of sugars promotes drought tolerance and
behave as a signal molecule in root growth of Azucena.
Sugars (trehalose and sucrose), N-containing molecules
(glycine and, betaine), and polyols were all identified as
suitable solvents for plant cells [46]. According to our
study, the sugar content of Azucena increased when
exposed to extreme water stress. Sugars (ribose, raffi-
nose, glucose, sucrose, xylonate, and erythronate) and
pyruvate, an intermediate of glycolysis, increased in
Azucena roots when water was scarce. These results are
consistent with a transcriptome analysis in potato and
barley after drought stress. A variety of genes involved in
Page 12 of 20
photosynthesis, sugar metabolism, flavonoid metabolism,
transporters, and transcription factors were found to be
involved in the drought tolerance response in plants sub-
jected to drought stress [47, 48]. Sucrose accumulates in
many plant tissues as a result of ecological stresses such
as water, salt, and cold stress [49].
Sugar alcohols, the primary photosynthetic products,
function as reserves of carbohydrate in many plants,
when water is scarce [50]. Mannitol has previously been
shown to promote tolerance to drought and salt, in trans-
genic tobacco and wheat [51, 52]. Compared with the
IR64, Azucena accumulated significant amounts of sugar
alcohol comprising of mannitol, sorbitol, myoinositol
in its roots, which could contribute to improved devel-
opment and promotion of the tolerance mechanism in
Azucena, while IR64 did not accumulate mannitol in
its roots. Proteomics and transcriptomics analysis of
drought stress in cucumber revealed that sorbitol and
mannitol are among the major players in plant adaptation
to salinity and drought stress [53]. Sorbitol and mannitol
penetrate the cell wall and alter the cellular response to
low water potential [54]. Ogbaga et  al. [55], studied the
response of two sorghums (Sorghum bicolor) cultivars
Samsorg 17 and Samsorg 40, to drought stress and found
substantial up-regulation of sugars (sedoheptulose, cello-
biose, fructose, galactose and lactose) and sugar alcohols
(ribitol, xylitol and myoinositol) in the tolerant samsorg
17. Trehalose is another metabolite that increased in
Azucena when exposed to drought stress. Trehalose is an
important osmolyte for osmotic control [56]. Trehalose
phosphate synthase 1 (TPS1) controls trehalose metab-
olism and has been demonstrated to increase drought
tolerance in a number of plant species [57]. Overexpres-
sion of the trehalose -6-phosphate phosphatase OsTPP3
increases drought tolerance in rice, transgenic plants
show higher root length in response to drought stress
[58]. Moreover, TPSP-transgenic plants exhibit less
sodium ion accumulation and less damage in roots under
salinity and drought stresses, and the transgenic seed-
lings also exhibited better growth as evidenced by greater
shoot and root length [59]. Furthermore, Deyanira et al.
2012 [60] reported that trehalose metabolism serves as
a biochemical marker in rice breeding because drought
increased trehalose synthesis only in drought-tolerant
varieties [60].
DAMs involved in amino acid metabolite and signal
sensing and transduction
Amino acids act as significant metabolites for synthesis
of protein and other cellular activities in plants; they also
maintain the osmotic potential of the cell, by acting as
osmolytes and also scavenge reactive oxygen species (ROS)
produced in drought-stressed plants [61]. According to one
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152
study, amino acids that encourage stomatal opening can
likewise undo the effects of ABA or mannitol on stoma-
tal closure, which lowers leaf evaporation [62]. Transcrip-
tomics and metabolomics profiling of drought-tolerant
and susceptible sesame genotypes, in response to drought
stress, showed that ABA, proline, arginine, lysine, aromatic
and branched chain amino acids, saccharopine, 2-aminoa-
dipate, and allantoin were higher in drought-tolerant gen-
otype under stress condition [63]. Valine, phenylalanine,
isoleucine, and tryptophan content consistently increased
under drought stress. Among them, tryptophan metabo-
lism was more increased in Azucena. L-tryptophan is a
physiological precursor to auxin and is crucial for reducing
the impacts of drought [64]. Drought tolerance was discov-
ered to be positively correlated with the expression level
of one of the tryptophan decarboxylase genes, which was
thought to be the initial enzyme gene for melatonin pro-
duction [65]. Additionally, tryptophan has only been found
to increase in wheat that is drought-tolerant [66]. Moreo-
ver, as tryptophan is involved in the metabolism of gly-
cine, serine, and threonine, it is probable that tryptophan
can increase plants’ ability to withstand drought. Under
conditions of water stress, Azucena accumulated greater
amounts of two different amino acids, fructoselysin and
L-cysteinylglycine, compared with IR64. L-cysteinylglycine
is a vital metabolite for numerous activities such as plant
growth, development, and response to drought stress due
to its unique biological properties, which are due to the
presence of Cys in the chemical reactivity of GSH (- SH)
and the higher water solubility of the thiol group [22]. This
L-cysteinylglycine with Cys and thiol can also be thought
of as a potent antioxidant in stress responses in drought-
resistant genotypes. Previous studies in Arabidopsis have
shown that oxidative stress increases protein glycation [56]
and both glucoselysin and fructoselysin exhibit antioxidant
activity [57]. The fructoselysin of the drought-tolerant gen-
otype could also function as an antioxidant, and is respon-
sible for drought tolerance. Isoleucine and phenylalanine
are two other amino acids found in large amounts in IR64.
Aside from their role in protein production, amino acids
also act as the building blocks for a number of other biolog-
ical processes and as precursors for a variety of secondary
metabolites. They are also crucial for antioxidant activity
and ROS scavenging. A low content of isoleucine helps the
plant to scavenge ROS [67]. However, in IR64, the content
of these amino acids is increased, which has a negative
effect on the scavenging activity of ROS, resulting in lower
drought tolerance of IR64.
GABA is essential for different functions, including car-
bon–nitrogen balance maintenance, pH control, and energy
synthesis [68], and it accumulates rapidly in stressed plants,
making it a stress marker. GABA content in the roots of
IR64 increased significantly during prolonged drought,
Page 13 of 20
whereas this metabolite did not appear in the metabolite
profile of the tolerant genotype. High GABA accumulation
in Arabidopsis restricts cell expansion and suppresses genes
involved in cell wall synthesis [69]. The accumulation of
GABA under drought stress conditions in barley might be
related to the suppression of primary root growth [70]. In
this study, it was observed that the accumulation of endog-
enous GABA not only restricted root development but also
reduced or delayed the formation of adventitious roots in
susceptible genotype.
Arginine can increase proline level [71], which was
reported to increase plant tolerance to stresses brought
on by freezing, salinity, and drought [72]. Proline was
the most significantly altered metabolite among all
common metabolites. Proline behaves as an osmolyte
for osmotic adjustment and helps in stabilization of
subcellular structures, prevention of oxidative burst
and is the explanation for increased drought toler-
ance [73]. Proline was found in higher concentrations
in the roots of Azucena under stress conditions than
in IR64 (39% more). Many plants have been shown to
be drought-resistant when proline accumulates, and
this accumulation is significantly larger in tolerant
genotypes than in sensitive genotypes [7]. It was pre-
viously found that higher concentrations of proline in
roots stimulate the synthesis of root hairs and root bio-
mass, resulting in vigorous plant growth despite poor
environmental conditions [16]. Due to its strong pro-
line accumulation, Azucena root can produce more
root biomass than IR64 under dry conditions. Proline
has several advantages: It acts as an osmolyte, contrib-
utes to the stability of cell structure, helps scavenge
free radicals and protect the redox potential of cells,
improves cytoplasmic acidosis, and maintains an opti-
mal NADP + or NADPH ratio in metabolism [7]. The
greater accumulation of sugars and amino acids in the
roots of the Azucena genotype may have promoted the
absorption of micro- and macro nutrients, resulting in
improved growth of root and thus more biomass. The
lower growth of genotype IR64 during drought could
be due to the synthesis of metabolites such as pyroglu-
tamic acid that were less responsive to drought, and
were produced in the roots only under stress. These
findings are congruent with previous studies that have
linked amino acids to plant root growth, symbiotic
partnerships, and diseases in the rhizosphere [74].
DAMs involved in TCA cycle and secondary metabolite
biosynthesis
It is well understood that pyrimidine and purine nucleo-
tides are essential for the production of lipids, peptides,
secondary metabolites and carbohydrates along with
being directly involved in the synthesis of nucleic acids
Ghorbanzadeh et al. BMC Genomics (2023) 24:152
[62]. Therefore, biosynthesis and metabolism of nucleo-
tides are of fundamental importance in the growth,
development and plant’s response to stress. Purine degra-
dation contributes to the protective responses to drought
stress, such as the buildup of the protective cellular
chemical proline and the synergistic activation of absci-
sic acid metabolism [22]. In the current study, Azucena
accumulated significantly more thymine (a purine nucle-
otide) and guanine (a pyrimidine nucleotide) compared
to IR64 [75]. Recent studies have shown that guanine and
thymine can contribute to drought tolerance of cereal
genotypes through energy production and enhancement
of defense responses [76].
Drought has been shown to elevate the concentra-
tion of phenolic compounds in various plants. Plants
form phenolic molecules (or polyphenols) as one of their
defense mechanisms against oxidative damage caused by
desiccation [77]. Some drought-resistant genotypes may
form more phenolic compounds with or without drought
treatment [38, 78, 79]. The total content of phenolics,
anthocyanins, saposides, and flavonoids in rice leaves
increased after drought treatment [80]. Earlier stud-
ies indicate that phenolic compounds have a protective
effect due to their unique structure, which includes dou-
ble carbon bonds, hydroxyl groups, and modifications
such as prenylation, methylation, and glycosylation [80,
81]. All the eight phenolic compounds found in Azucena
in this study had hydroxyl groups, double carbon bonds,
or methyl groups. Consequently, the phenolic compo-
nents of Azucena could behave as potent antioxidants
and thus prevent the oxidative damage caused in plants
by drought stress.
Drought tolerance of plant species is affected by a
change in the content of various organic compounds
due to environmental stress conditions. The compara-
tive content of organic matter depends significantly on
the specific metabolites under the given conditions, such
as succinic acid, malic acid, and galacturonic acid, which
show the largest increases of all organic acids in response
to long-term drought stress. Levi et  al. [82] observed
that increase of various organic acids, particularly citric
acid, could lend to improved drought tolerance in some
cotton genotypes. Malic acid increased drought toler-
ance in several plant species including speargrass, cot-
ton, and tropical grasses [83, 84]. In the root, Azucena
accumulated 3-dimethylallyl-4-hydroxymandelic acid,
4-hydroxy-2-oxoglutaric acid, succinic acid, salicylic
acid, and isocitric acid to a considerable extent; in con-
trast, IR64 accumulated only phosphoric acid in higher
concentrations. Another metabolite, 4-hydroxy-2-oxog-
lutaric acid, increased five-fold in Azucena roots during
drought stress. In another study, 4-hydroxy-2-oxoglutaric
acid was not detected in N. tabacum leaves, but in N.
Page 14 of 20
tabacum roots, a 20-fold increase was detected in the
first hours (between 1 and 2  h) and a 70-fold increase
after four hours of drought stress. This indicates that N.
tabacum stores 4-hydroxy-2-oxoglutaric acid as soon as
water is not available, which is then reduced to pyruvate
and glyoxylate [80]. Isocitric acid, a constituent of the
TCA cycle, was deposited at considerably higher levels
in the tolerant genotype roots, which may have contrib-
uted to the maintenance of a vigorous root system and
assimilation of nutrients and water in the current study.
The TCA cycle serves as both an energy source and a car-
bon scaffold for the production of various amino acids
[85]. The obtainability of carbon and the energy that is
required for cell division influence the development of
roots [86]. TCA cycle intermediates such as citric acid,
malic acid and fumaric acid have been shown to be
higher in stressed roots [87].
DAMs involved in lipids and fatty acid metabolites
biosynthesis
Fatty acids are important for several biological processes,
including energy production and the formation of mem-
brane lipids in living organisms. Plant defense systems
also rely on fatty acid metabolic pathways [88]. Changes
in lipid profiles brought on by stress cause membrane
lipid remodeling and activate plant defense mecha-
nisms in response to biotic and abiotic stresses, such as
drought [89, 90]. The amount of alpha-linolenic acid in
the stressed roots of the Azucena genotype was increased
when compared to the control condition. Moreover, the
content of alpha-linolenic acid under well-watered condi-
tions was significantly higher in the Azucena compared
to IR64, showing a genotype-specific expression pat-
tern. Thus, the increased metabolism of linolenic acid in
drought-stressed plants is consistent with the intrinsic
drought tolerance of the Azucena genotype. Our results
indicate that Azucena can be employed as an important
genetic resource to improve drought tolerance in other
cereals. According to Gundaraniya and Ambalam [91],
the buildup of saturated FAs (stearic acid) was enhanced
in the leaves of a drought-tolerant peanut genotype dur-
ing drought stress, while the accumulation of 8,11-octa-
decadienoic acid decreased in the roots of a peanut
genotype, that is drought-sensitive.
Profiling of genes involved in drought stress response
In addition to metabolite profiling, qRT-PCR was exe-
cuted to validate some of the major genes involved in
the root growth and elongation. Our results indicated
that the expression of DREB2B and IAA19 were much
higher in the drought tolerance genotype. Auxin co-
receptors and transcriptional repressors known as Aux/
IAA proteins are essential for auxin signaling in plants.
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152
GLS content is controlled by IAA19, IAA6, and IAA5,
the auxin-responsive Aux/IAA repressors. These pro-
teins maintain high GLS expression in drought-stressed
plants through a transcriptional cascade [92]. These Aux/
IAA proteins are produced by the Aux/IAA genes IAA19,
IAA6, and IAA5, which are directly controlled by the
drought-responsive transcription factors DREB2A and
DREB2B [93]. Synthesis, transport and signal transduc-
tion of auxin are essential for root elongation and adven-
titious root development [94]. IAA is formed via the
Trp-dependent or Trp-independent pathway. Trp bio-
synthesis is considered an important step in both path-
ways [95]. Auxin is synthesized from Tryptophan via the
shikimic acid pathway [96]. It has been previously shown
that an increase in auxin levels promotes shoot and root
development in a mixed nitrogen environment [87]. The
observed changes in the stress-dependent formation of
quinic acid and shikimic acid could be a cause of root
elongation.
Conclusions
The cell and tissue structure of plants is affected by
drought stress. Therefore, a multidisciplinary approach
(genomics, transcriptomics, proteomics, and metabo-
lomics) would increase our knowledge of the funda-
mental processes of drought tolerance in rice. GC–MS
Metabolomics was applied to differentiate root tip
metabolites under stress and well-watered conditions.
This study showed that roots from two varying genotypes
subjected to drought stress, had different mechanisms
for regulation and accumulation of metabolites, which
is important for understanding the overall mechanisms
of drought stress tolerance. Moreover, there was signifi-
cant change in 103 metabolites of the root tips during
drought stress, and the concentration of most metabo-
lites increased. To maintain osmotic balance, the con-
centration of various compatible solutes such as sugars
and polyols was increased. Positive correlations between
metabolite content and drought tolerance characteris-
tics were found for trehalose, proline, sucrose, succinic
acid, aspartic acid, tryptophan, salicylic acid and manni-
tol. The concentrations of these metabolites were much
higher in tolerant plants than in sensitive plants. Under
water-limited conditions, Azucena performed better
than IR64 because the root elongation rate increased
more in this genotype, which could be due to the promo-
tion of the TCA cycle in this genotype. Under drought
conditions, the biosynthetic pathways of several amino
acids, such as alanine, glutamate, and aspartate, was
increased in the roots of Azucena and IR64. Based on the
metabolomic and phenotypic changes in the roots, such
as greater swelling capacity, faster transport from source
to sink, and better absorption capacity, Azucena could
Page 15 of 20
be a promising genotype for future drought breeding
programs.
Materials and methods
Plant material and stress treatment
IRRI provided the seeds of two known contrasting rice
genotypes (Oryza sativa L.), Azucena (a deep-rooted
upland Japonica-type genotype), and IR64 (a shallow-
rooted lowland Indica-type genotype) from the Interna-
tional Rice Genebank Collection. Seeds were germinated
on wet filter paper after sterilization. Then, the 7-day-
old, equal-sized seedlings were hydroponically grown in
Yoshida solution at 22–25  °C, relative humidity of 85%,
and a 16 h light/8 h dark photoperiod for two weeks [97].
On the 20th day, the seedlings were transferred into root
boxes filled with 1:1:2 combinations of sand, peat, and
clay. The root boxes were constructed from 5  mm thick
Plexiglass sheets and then transferred to the greenhouse
(with the same environmental conditions). Drought
stress was imposed upon the 35-day-old well-watered
plants by withholding irrigation for 14  days until field
capacity reached 25%-35%. Control plants were irrigated
regularly. According to Puértolas [98], field capacity was
randomly determined twice daily for at least 40 root
boxes during the treatment period. Using a Soil Moisture
Sensor SM150, the soil moisture of the 40 root boxes was
randomly tested (Delta-T Devices, UK). The collection
of seeds and the complete experiment was carried out
according to the national guidelines [99].
Root morphological parameters assay
After 16  days of treatment, complete roots from three
plants of each genotype were collected, washed, and
dried to analyze root morphological characteristics
under control and stress. The SmartRoot system (https://​
www.​quant​itati​ve-​plant.​org/​softw​are/​smart​root) was
used to measure average root diameter, length and num-
ber of lateral roots, root length, number of tips, surface
area, number of branches, and overlap sections (intersec-
tions) [100, 101]. The diameter of the primary and lateral
roots was determined under an optical light microscope
(Olympus BX51, 10X objective).
Sampling and metabolite extraction
Root tip sections from about 3 plants were pooled as bio-
logical replicate, and eight independent biological rep-
licates were taken. Freshly collected root-tip samples of
Azucena and IR64 genotypes were pulverized thoroughly
using liquid nitrogen and mortar and pestle, and about
100  mg of this powder was taken in a 5  ml centrifuge
tube. 1400 μL of pre-chilled methanol was then added
to each tube and shaken for about 30  s before 60 μL of
Ghorbanzadeh et al. BMC Genomics (2023) 24:152
ribitol (0.2  mg  ­mL−1) was supplemented as an internal
quantitative control and shaken for a further 30  s [24,
102]. The tubes were then placed in an ultrasonic appa-
ratus for 30 min at room temperature (25 °C), after which
750 μL and 1400 μL of pre-chilled chloroform and deion-
ized water (dH2O), respectively, were supplemented
and vortexed for a minute, followed by centrifugation at
14,000 r ­min−1 (4 °C) for 10 min. One ml of the superna-
tant was then transferred into a new tube; vacuum dried
in a concentrator and then 60 μL of Methoxyamine-
pyridine (15  mg  ­mL−1) solution was added, vortexed
(0.5 min), and allowed to react at 37 °C for two hours. To
this 60 μL of the BSTFA reagent was added and the mix-
ture was incubated for 1.5 h at 37 °C and then centrifuged
at 12,000 r ­min−1 (4 °C) for 10 min and the supernatant
was placed in a vial for GC–MS analysis [103].
Untargeted metabolomics analysis
Non-targeted metabolite profiling was carried out using
gas chromatography-mass spectrometry (GC-TOF–MS),
according to Ghaffari et  al. 2016 [104]. The injection of
1μL of each sample was done using a split ratio of 10:1
into a GC–MS, Shimadzu QP2010Plus, Japan) equipped
with a DB-17 MS capillary column (0.25 mm I.D., 30 m
length, 0.25  μm film thickness; Agilent Technologies
Inc) at 1.2 mL ­min−1 constant flow rate. The samples in
replication were continuously injected in random order
to discriminate the technical variations from biological
ones, as one batch. The temperatures for the ion source,
the transfer line and the injector were set to 230  °C,
280  °C, and 280  °C respectively. Helium (purity 99.99%)
was used as the carrier gas, flowing at a rate of 1 mL per
minute. A 70  eV electron ionization was utilized in the
full scan mode (50 − 1000  Da, m/z). The mass spectra
provided the result data files in CDF formats, which were
then imported into the XCMS software using the R soft-
ware platform (http://​cran.r-​proje​ct.​org). XCMS could be
automatically preprocessed using procedures such peak
detection, data baseline filtering, raw sign al extraction,
and integration [105]. Ultimately, after alignment using
the statistical component for comparison, the ‘CSV’ file
was acquired which included data sets like retention
time, sample information, and intensity of peaks. For
quality control of the data (reproducibility), the internal
standard ribitol was used. Additionally, all false posi-
tive peaks, such as those brought on by column bleed,
noise, the BSTFA derivatization procedure, and inter-
nal standards, were eliminated from the data set. The
total peak intensity of each sample was used for data set
normalization, after which each sample was separately
imported into the SIMCA-P software package. (Version
11.0, http://​www.​umetr​ics.​com/​simca). The untargeted
Page 16 of 20
metabolite data was processed, and the online statistical
software MetaboAnalyst 5.0 (https://​www.​metab​oanal​
yst.​ca/) [106] was used for statistical analysis. The peak
areas of the chromatograms were considered for statisti-
cal analyses.
The search program database National Institute of
Standards and Technology, Gaithersburg, MD, USA
(NIST) [107], the GOLM metabolome database (GMD)
[108], and the Wiley Registry of Mass Spectral Library
were updated with structural information and thus
allowed the discovery of new and targeted metabolites.
All matched spectra were analyzed and verified with
authentic standards [109]. In the studies, peaks of the
metabolites with more than 70% similarity index were
considered effective, while those with less than 70%
were deleted from the data [110]. Each metabolic com-
ponent was assigned its own lane for measurement and
contained amino acids, organic acids, and sugars [111].
The peak areas were normalized to the area of a single
lane of the internal standard, giving the relative reac-
tion ratios normalized by the fresh weight of each sample
­(log2-transformed).
Metabolomics data processing and analysis
Significant analysis of metabolites (SAM) and principal
component analysis (PCA) were individually utilized to
pinpoint the most significant metabolites that get altered
in the different genotypes during stress [112]. To identify
the metabolites that were significantly changed between
different conditions (control and drought), or in different
genotypes (tolerant and sensitive) (P value < 0.01), mul-
tifactorial analysis of variance (ANOVA) was executed.
Additionally, Partial Least Squares Discriminant Analysis
(PLS-DA) was used in this work to optimize and identify
differences between the metabolic profiles of the control
and drought-stressed plants. The R2 and Q2 (goodness
of prediction) values, which reflected the total explained
variance and the model predictability, were collected
from these studies using a default seven-fold internal
cross-validation. Pathway analysis was implemented
for better interpretation of the functions of the altered
(significantly changed at P < 0.01) metabolites using the
MetaboAnalyst 5.0 (https://​www.​metab​oanal​yst.​ca)
[113], via Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway database (http://​www.​genome.​ad.​jp/​
kegg/​pathw​ay.​html) and compared with Oryza sativa
ssp. japonica (Rice Annotation Project Data Base http://​
rapdb.​dna.​affrc.​go.​jp) pathway library [114]. The t-test
(P-value < 0.05) was performed to assess the statistical
significance of the root morphology data [113].
 Ghorbanzadeh et al. BMC Genomics (2023) 24:152
Correlation analysis
Correlation analysis between metabolite levels, root
phenotyping and candidate gene expression data sets of
paired samples was performed using Pearson correla-
tions (function cor. test, R) according to Ghaffari et  al.
2016 [104].
Quantitative gene expression analysis
Consistent with our previous miRNA-seq and mRNA
reports [31, 32], 13 genes involved in root growth and
elongation and regulation of root primary metabolism
were selected. Total RNA isolation was done using the
TRIzol reagent (BioBasic-BS410A, Canada) as per the
manufacturer’s guidelines. RNA quality was assessed
NanoPhotometer® spectrophotometer (NP80 NanoPho-
on an agarose gel (1%). RNA quantity was found using a
tometer, IMPLEN). The cDNA was reverse transcribed
from the isolated RNA using the SuperScript First-Strand
Invitrogen™ Kit. PerlPrimer v.1.1.21 software was used
System for the RT-qPCR, which was performed using
for primer designing from the transcribed region of the
rice genes (sequences obtained from the RAP-DB data-
base). qRT-PCR was accomplished using SYBR Green
Master Mix (Eurogentec, Köln, Germany) in the ABI
Prism 7900HT (Applied Biosystems, Foster City, CA)
with the usual thermal cycling conditions (95  °C for
10 min, 95 °C for 15 s for 40 cycles, 60 °C for 1 min). The
experiment was conducted in three biological replicates
and two technical replicates were used. For the purpose
of examining the dissociation curves for shoulders or
extra peaks, the SDS 2.2.1 software (Applied Biosystems)
was utilized. The expression values were normalized
using the UBQ (Ubiquitin) gene as a housekeeping gene
[115]. LinRegPCR was used to calculate primer efficiency
[116]. “Normalized expression of the genes of interest
was calculated by dividing the average relative expression
(primer efficiency P to the power of cycle number Ct) of
the housekeeping genes (H1) by the relative expression
of the gene of interest (GOI): (GOI): ((PH1^CtH1)/2)/
PGOI^CtGOI.”
Abbreviations
FC Field capacity
GC–MS Gas chromatography-mass spectrometry
PCA Principal component analysis
PCs Principal components
PLSDA Partial least-squares-discriminant analysis
VIP Variable importance in the projection
QTL Quantitative trait locus
RL Root length
SA Surface area
NIST National Institute of Standards and Technology
GMD GOLM metabolome database
SAM Significant Analysis of Metabolites
cDNA Complementary DNA
Page 17 of 20
DAMs Differentially abundant metabolite
OsTPP1 Trehalose -phosphate phosphate
SUT5 SUCROSE TRANSPORTER 5
DREB Dehydration-responsive element binding protein
PRP5 Proline-rich protein
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12864-​023-​09246-z.
Additional file 1: Table S1. Identification results of differently accu-
mulated metabolites between two rice genotypes under drought.
Table S2. List of significant metabolites (between normal and drought
conditions) identified through ANOVA with their p-value in root tip IR64.
Table S3. List of significant metabolites (between normal and drought
conditions) identified through ANOVA with their p-value in root tip Azu-
cena. Table S4. The KEGG pathways  of the altered metabolites exposure
to drought stress in Azucena root samples. Table S5. The KEGG pathways
of the altered metabolites exposure to drought stress in IR64  root
samples. Table S6. List of primers with details of sequence and expression
profile used for RT-qPCR validation.
Acknowledgements
The authors thank the Agricultural Biotechnology Research Institute of Iran
(ABRII) for providing lab facilities for this work.
Authors’ contributions
ZGH collected samples, performed laboratory procedures, prepared the first
draft of the manuscript, and performed data analysis and figure prepara-
tion. RH assisted with data analysis, and figure preparation, and edited the
manuscript. FJ and MZ assisted in editing the manuscript. GHS edited the
manuscript and provided supervision. MRG conceptualized the experiments,
data analysis, editing of the manuscript, and supervision. The author(s) read
and approved the final manuscript.
Funding
This research was not supported by any agency.
Availability of data and materials
The original contributions presented in the study are included in the article/
Supplementary Material; further inquiries can be directed to the correspond-
ing authors.
Declarations
Ethics approval and consent to participate
All experimental studies on plants were complied with relevant institutional,
national, and international guidelines and legislation.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
 Department of Systems Biology, Agricultural Biotechnology Research Insti-
tute of Iran (ABRII), Agricultural Research Education and Extension Organiza-
tion (AREEO), Karaj, Iran. 2 Department of Plant Breeding, Cotton Research Insti-
tute of Iran (CRII), Agricultural Research, Education and Extension Organization
(AREEO), Gorgan, Iran. 3 Centre for Plant Biotechnology and Molecular Biology,
Kerala Agricultural University, Thrissur, India. 4 Department of Molecular Sci-
ences, Macquarie University, North Ryde, NSW, Australia.
Received: 7 September 2022 Accepted: 14 March 2023
Ghorbanzadeh et al. BMC Genomics (2023) 24:152
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