Pak. J. Bot., 56(4): 1209-1224, 2024. DOI: http://dx.doi.org/10.

30848/PJB2024-4(22)

RIBOFLAVIN (VITAMIN B2) PRIMING MODULATES GROWTH, PHYSIOLOGICAL

AND BIOCHEMICAL TRAITS OF MAIZE (ZEA MAYS L.) UNDER SALT STRESS
NOSHEEN IFTIKHAR1 AND SHAGUFTA PERVEEN1*
1
Department of Botany, Government College University, Faisalabad-38000, Pakistan
*Corresponding author's email: perveens1@yahoo.com

Abstract

Abiotic stresses are more often to occur because of poor drainage system resulting in agricultural land contaminations.
Salt stress is one of the abiotic stresses which highly effects the growth and yield of cereal crops especially maize (Zea mays
L.). The current experiment following completely randomized design (CRD) along with three replicates of each treatment was
performed in the Botanic Garden, Government College University, Faisalabad to evaluate the effect of salt (70 mM NaCl)
stress on maize (Zea mays L.) plants raised from seeds treated with riboflavin (RF). The temperature range in whole experiment
was 30°C - 43°C. This study appraised riboflavin (0, 50 and 75 ppm) role in stress effect mitigation by enhancing growth,
enzymatic (SOD, POD, CAT, APX and GPX) and non-enzymatic (flavonoids, phenolics and anthocyanin) antioxidant
activities, and by scavenging ROS (MDA, H2O2) effect and maintaining osmotic level. Salt (70 mM NaCl) subjected plants
showed reduced growth and photosynthetic rate, while increased ROS production (more in Sadaf compared to Pearl).
Riboflavin is a novel vitamin which can be used to treat the salinity stress effected plants. Seed priming with RF (vitamin B2)
significantly reduced salt stress effects by enhancing growth rate, photosynthesis, increased osmolytes accumulation and
improved antioxidant defense system, while decreasing oxidative stress (MDA and hydrogen peroxides). Plants raised from
seeds treated with riboflavin showed a significant increase in total leaf area, total free proteins and total soluble sugars than
plants without riboflavin application. Gradual increase in RF concentration showed more improved growth under salt stress.

Key words: Antioxidants, Maize, Priming, Riboflavin, Salinity.

Abbreviations: RL- Root length, SL- Shoot length, RFW- Root fresh weight, RDW- Root dry weight, SFW- Shoot fresh weight, SDW-
Shoot dry weight, LA- Leaf area, RWC- Relative water contents, Chl.- Chlorophyll, T. Chl.- Total Chlorophyll, MDA- Malondialdehyde,
Antho.- Anthocyanin, Flavo.- Flavonoid, Phenol.- Phenolics, Pro.- Proline, T.Pro.- Total proteins, TSS- Total soluble sugars, SOD-
Superoxidase dismutase, POD- Peroxidase, Cat.- Catalase, APX- Ascorbate peroxidase, GPX- Guaiacol peroxidase, RF- Riboflavin

Introduction effect (Hasanuzzaman et al., 2020). Soil salinity stimulate

activities of superoxide dismutase, peroxidase, ascorbate
Soil salinity is the global issue, threating the food peroxidase and catalase enzymes to mitigate stress effects
production and environmental health by affecting about (Lee et al., 2001).
954 million hectare of land worlwide (Diaz et al., 2021; Maize (Zea mays L.), a member of family Poaceae, is
Wei et al., 2021). Increased salinity concentration of either the 2nd most demandingly produced cereal crop worldwide
rootzone or surface area decreases the soil fertility rate and (Santpoort, 2020; Vaughan et al., 2018). It is known as the
ultimately decreases plants production rate (Fu et al., king of cereal crops. It is produced in 116 countries all over
2020). This salinity hinders water intake and results in the world (Ahmad et al., 2021; Waqas et al., 2021).
erosion (Gorji et al., 2020) and effects agricultural growth Environmental changes have a deleterious effect on Z.
and decreased crop production (Sing, 2022). mays growth and yield ultimately decreasing the food
Salinity is threating abiotic factor which limits the availability and leading to corn deficiency (Wichelns &
growth, development, yield and expansion of plants (Zhao Qadir, 2015). There is a reduced production rate of Z. mays
et al., 2020, Hadia et al.,2022). Salinity stress induce and it looks hard to increase its production rate to fulfil the
several morphophysiological changes that results in deficiencies (Zahra et al., 2020; Kaya et al., 2020).
decreased shoot and root lengths, fresh and dry weight of Vitamins have ability to protect plants from singlet
plant (Dikobe et al.,2021). It is the main growth reducing oxygen and hygrogen peroxide species which are produced
factor which delays the germination time and causes during photosynthetic process. Vitamins are important for the
disruption in nutrients uptake resulting in plant nutrient improvement of photosynthetic rate and antioxidant enzymes
deficiency (Shiade & Boelt, 2020). Soil salinity subjected activities (Chi et al., 2021). These are also important for any
plants showed reduced Chl. a, b and total chlorophyll plant for their growth, yield production and metabolism
contents. Increased soluble sugar accumulation is followed improvement. These may be fat or water-soluble (Garg et al.,
by decreased starch level (Hassanein et al., 2002; Waheed 2021). Many biological responses are highly affected by
AL-Mayahi, 2016). Salt stress causes accumulation of vitamins and vitamin B2 is highly produced in green leaves of
hydroxyl radical and hydrogen peroxide which is signal of vegetables (Yoshii et al., 2019). Vitamins are important for
oxidative stress in plants that might be the reason of cell competing the environmental biotic and abiotic stresses,
damage and if there is no appropriate defence mechanism; especially vitamin B are the base of metabolic cofactors but
cell dies. So plant’s antioxidative machinery is activated to any harsh stress level may cause decrease in B group vitamins
mitigate stress effects (Nadarajah, 2020; Abd El-Samad et in plants. To mitigate such conditions exogenous vitamin
al., 2017). Both enzymatic and non-enzymatic antioxidants application is practised (Abdulhamed et al., 2020). Seed
help the plants to detoxify reactive oxygen species (ROS) soaking technique is one of the best treatments for seeds to
1210 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

compete against stress conditions by adapting physiological Chlorophyll contents: For the determination of
improvements (Hadia et al., 2022). This is a cost effective chlorophyll contents the protocol of Arnon (1949) was
technique and give great results. This technique positively used. Fresh leaves (0.5 g) were taken, chopped and ground
increases seed maturation and plant growth rate (Kazemi & in 10 ml of 80% acetone (20 ml water and 80 ml acetone)
Eskandari, 2012; Hafeez et al., 2021). and left these at -4oC for one night then centrifuged these
Riboflavin is known as a water-soluble vitamin B2 samples at 10,000 rpm for 5 minutes. Then the supernatant
(Jiadkong et al., 2023). Its importance in every field either was used to measure chlorophyll contents at wavelengths
food production or medicine is dramatically increased due of 480 nm, 645 nm and 663 nm through a
to its participation in health nutrition (Zhou et al., 2021). spectrophotometer (IRMECO U2020).
Riboflavin due to its anti-oxidative characteristic plays an
important role in competing the salinity effect by The following formula was used to measure chl. a, chl.
improving osmotic pressure and ultimately enhances b contents.
abiotic stress resistance (Abdulhamed et al., 2020; Singh,
2022). When plants are exposed to salinity stress, Chl. a = [12.7 (OD 663) -2.69 (OD 645)] × V/1000 × W
exogenous vitamin application activate antioxidant defense
system that increase stress tolerance in maize, tomato and Chl. b = [22.9 (OD 645) -4.68 (OD 663)] × V/1000 × W
rice plants (Alayafi, 2020, Khatun et al., 2016).
In order to check the role of riboflavin (vitamins B 2) V = Volume of the extract (mL),
in decreasing salt stress effects; a pot experiment was
performed, where two salt stressed maize cultivars were W = Weight of the fresh leaf tissue (g)
treated with riboflavin.
Malondialdehyde (MDA) contents: Malondialdehyde
Experimental Design: A sand pot (8L) experiment was (MDA) contents were estimated by following the protocol
conducted in the Botanic Garden, Government College of Cakmak & Horst (1991). To 0.5 g fresh leaf material
University, Faisalabad. Day to night humidity was 61%- added 10 ml of 0.1% w/v TCA (trichloroacetic acid) during
70% and day to night temperature was 30°C-43°C. Maize grinding. Then centrifuged this solution at 12,000 rpm for
(Zea mays L.) seeds of two varieties (Sadaf, Pearl) were 10 minutes and took 1 ml of extract and to it added 4.5 ml
collected from Ayub Agricultural Research Institute of 0.5% TBA (thiobarbituric acid). The mixture was heated
Faisalabad and Maize and Millet Research Institute, in a water bath for 30 minutes at 95oC, cooled in an ice bath
Sahiwal. A completely randomized design (CRD) was and again centrifuged. The readings of above samples were
followed and two salt stress levels (0 and 70 Mm NaCl) observed at 532 nm and 600 nm wavelengths on a
were used to check the effect of salt stress. After gentle spectrophotometer (IRMECO U2020).
wash seeds of both varieties were primed with three
riboflavin (vitamin B2) levels (0, 50 and 75 ppm) for 12 Hydrogen peroxide (H 2O2): For the estimation of
hours and seed sowing was done in form of six hydrogen peroxide contents method purposed by
experimental sets. Velikova et al., (2000) was used. Leaf sample of 0.5 gram
was ground by adding 5 ml of 0.1% w/v trichloroacetic
Sampling and data collection: After uniform acid (TCA). Mixture was centrifuged at 12,000 rpm for
germination, at four-leaf stage, salt stress was applied 15 minutes. Phosphate buffer of neutral pH (7.0) was
along with full strength Hoagland’s nutrient solution. added with 0.5ml concentration after that 1ml of KI
Hoagland’s solution was applied every 3 rd day throughout (potassium Iodide) was added and absorbance was noted
the experiment. During the fourth week of germination, at 390 nm wavelength on UV visible spectrophotometer
plants were uprooted, well washed and air dried to wipe out (IRMECO U2020).
excessive water. Root and shoot fresh weights and lengths
were measured. After that shoot and root of each plant Anthocyanin contents: For anthocyanin estimation
sample were placed in an oven for 48 hours at 72oC for dry method of Zhang et al., (2009) was followed. According
mass measurement. Total Leaf area per plant was to this method fresh leaves (0.1 g) were ground in 5 ml of
calculated using the method of Carleton & Foote (1965). phosphate buffer and then centrifuged. Values of samples
For the determination of physiological and biochemical were noted at 600 nm with a spectrophotometer
parameters, leaves were packed in plastic zipper bags and (IRMECO U2020).
kept in freezer at -20oC.
Flavonoids: For flavonoids determination protocol of
Plant analysis Karadeniz et al., (2005) was used, fresh leaves (1.0 g) were
taken from each of the plants and then ground with a pestle
Relative water contents (RWC) (%): Fresh leaves of and mortar by adding 20 ml of 80% ethanol. Samples were
each plant were taken and weighed. Then, soaked the filtered through Whatman,s filter paper 42. In a test tube
leaves in de-ionized water for 24 hours. Weight of soaked 0.5 ml filtrate and 3 ml deionized water along with 3 ml of
leaves was measured and these leaves were placed in an 0.5% NaNO2 and 0.6 ml of 10% AlCl3 was also added and
oven at 80oC for 48 hours. left the samples for 6 minutes. Added 2ml of 1M NaOH.
And dry weight was measured (Jones & Turner, 1987). Deionized water was added to make the volume of each
test tube up to 10 ml. The reading of flavonoid at 510 nm
RWC% = [(FW - DW)/ (TW - DW)] × 100 was noted with a spectrophotometer (IRMECO U2020).
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1211

Phenolic content: Total phenolic contents were determined Peroxidase (POD) and catalase (CAT): Activities of both
by the protocol of Julkunen-Titto (1985). Leaf sample (0.05 POD and CAT were determined by following the method
g) of each replicate was taken and ground with 10 ml of 80% of Chance & Maehly (1955). For CAT determination, a
acetone. After homogenizing the leaf sample with acetone, mixture was prepared to consist of 1.9 mL (50 mM) with
it was centrifuged at 10,000 rpm for ten minutes. Removed pH 7, 5.9 mM of hydrogen peroxide (1 mL) and 100 µM
supernatant and a little portion of aliquot (100 µ) were of enzyme extract. Readings were noted at 240 nm on a
treated with 1 ml of Folin-Ciocalteau’s phenol reagent and spectrophotometer. Readings were monitored every 20s for
then 2.0 ml of distilled water was added. After that, 5 ml of 2 minutes. While for POD estimation, a reaction mixture
Na2CO3 (20% w/v) was added. Deionized water was added was prepared to contain 250 µL (50 mM) of phosphate
to make the volume up to 10 ml, solution was shaken and the buffer, 100 µL (20 mM) guaiacol, 50 µL enzyme extract
values were observed at 750 nm on UV visible
and distilled water. The enzyme activity change was
spectrophotometer (IRMECO U2020).
observed every 20s at 470 nm.
Free proline estimation: Free proline contents were
estimated by the method given by Bates et al., (1973). A Ascorbate peroxidase (APX): A reaction solution (3mL)
fresh leaf sample (0.5g) was taken and ground in 10mL of which was consisting of 50 mM phosphate buffer with 7.0
sulphosalyslic acid (3%) and was left for 5 minutes. Filtered pH, 0.5 mM hydrogen peroxide and 0.5 mM ascorbic acid
it, 2mL of filtrate, 2mL acid ninhydrin and 2 ml of glacial was prepared (Asada, 1992). The reaction was started after
acetic acid were poured into the test tube and heated at 100oC adding hydrogen peroxide. The absorbance was recorded
for one hour. The sample was cooled in an ice bar. Then 4mL at 390 nm for 2 minutes.
of toluene was added, stirred and passed through the air for
2 minutes. The values were observed at 520 nm absorbance. Guaiacol peroxidase (GPX): The determination of GPX
Toulene was run as blank. Proline concentration was was done by using (3 mL) guaiacol solution containing 10
observed through the standard curve. mm (K2H2PO4) at 7 pH, 20 mm guaiacol, 0.5 mL crude
extract and 20 mm guaiacol and values were noted at 436
Total soluble Proteins: Total soluble proteins were nm (Chance & Maehly, 1955).
determined by the protocol given by Bradford (1976).
Fresh leaves (0.5g) were well ground in 10 mL (50 mM) Acid digestion for ion analysis (Allen et al., 1985): The
phosphate buffer in a prechilled environment, then dried material (0.1 g) was placed in a digestion flask and 2
centrifuged at 6000 rpm for five minutes at 4°C. 0.1 mL of ml of sulphuric acid (H2SO4) was added. The mixture was
supernatant and 2mL of Bradford reagent were introduced left for over night at 25oC. On the next day, 0.5ml of
and this mixture was left for five minutes. Absorbance was hydrogen peroxide was added and heated at 150 oC. then
noted at 595 nm absorbance using a spectrophotometer. placed the flasks on hot plate at 250oC temperature. The
fumes emission and coloration were observed. When the
Total soluble sugars: Total soluble sugars were solution turned colorless, volume of the solution was up to
determined by the method of Yemm & Willis (1954). Dried 50 ml by adding water. It was filtered and was run on flame
plant material was ground and passed through sieves of photometer for the determination of ions (K, Ca and Na).
1mm. The extracted material (0.1 gram) was mixed with Cl- was determined by AgCl precipitation and titration
80% acetone (10 mL each), shaken for 6 hours and the procedure following the method of Johnson & Ulrich (1959).
extract was used for the determination of soluble sugars,
0.1 mL plant extract and 3mL of anthrone reagent were Statistical Analysis
poured into a 25 mL test tube. Then, heated at boiling
temperature for 10 minutes and was cooled it for 10 The collected data was subjected to analysis of
minutes. Incubated for 20 minutes at room temperature and variance (ANOVA) using CoStat software version 6.303
absorbance was noted at 625 nm using a spectrophotometer and mean values were compared by least significant
(IRMECO U2020). difference (LSD) test at 5% level of significance.

Determination of antioxidant enzymes activities: Under Results

super chilled conditions, 0.5 gram of fresh leaves were ground
in 10 ml (5 mM) phosphate buffer with 7.8 pH, centrifuged at Growth traits: The current experiment was conducted to
12000 rpm at 4°C (20 min) and again centrifuged this at 15000 check the effect of seed priming with riboflavin (RF) i.e.,
rpm for 10 minutes. Stored this enzyme extract at -20°C for 0 ppm, 50 ppm and 75 ppm on two maize (Zea mays L.)
antioxidant enzyme activity analysis. varieties (Sadaf and Pearl) under two levels of salt (0 and
70 mM NaCl) stress. Root and shoot length of Sadaf
Superoxide dismutase (SOD): Enzyme inhibition of showed more reduction than Pearl under salt stress (70
photochemical reduction of nitroblue tetrazolium (NTB) mM). Shoot length showed comparatively more reduction
was determined by the method of Giannopolitis & Ries rate than root length (p≤0.001) and pre-treatment with
(1977). For this purpose, a reaction mixture was prepared. riboflavin exhibited improved plant (p≤0.01) response
This mixture contained 250µL (50 mM) of phosphate under salt stress. Highly reduced root and shoot fresh
buffer, 400µL distilled water, Methionine (100µL), 50 µM weight was observed in the variety Sadaf than that of Pearl.
NBT, 50 µM riboflavin and 50µL of enzyme extract. Then However, this reduction was less in plants raised from the
values were noted at 560 nm absorbance on UV visible grains treated with RF. The dry mass of Sadaf was less than
spectrophotometer. that of Pearl. Variety Sadaf showed high (p≤0.05) reduction
1212 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

rate in dry mass under salt stress and RF treated plants were Antioxidant enzymes activities: Activities of antioxidant
significantly tolerant to salt stress (p≤0.01). The Sadaf enzymes i.e., SOD, POD, CAT, APX and GPX
variety has a greater response under salt stress (p≤0.01) in significantly increased under salt stress in both maize
terms of leaf area, however, RF treated plants exhibited a varieties (Pearl and Sadaf). Thus, Pearl was higher in the
less salt stress effect (p≤0.05). Overall, all growth attributes activities of antioxidant enzymes compared to Sadaf
i.e., root and shoot fresh and dry weights, shoot and root variety. Pre-sowing seed treatment with riboflavin further
lengths, and total leaf area per plant (p≤0.001) have enhanced the activities of antioxidant enzymes. Riboflavin
decreased under salt stress, while pre-sowing seed played its significant role in the improvement of
treatment with riboflavin improved these growth attributes antioxidants activation rate (p≤0.001) (Table 1; Fig. 3).
of both maize varieties under salt stress or non-stress
conditions. Pearl was higher in growth parameters than Ion analysis: Salt (NaCl) stress increased Na+ and Cl- ions
Sadaf variety of maize (Fig. 1, Table 1).
in root and shoot of both varieties, however, its
enhancement rate was greater in Sadaf variety than that of
Relative water contents (RWC) and chlorophyll
Pearl. Salt stress decreased nutrient ions (K+, Ca+) in both
contents: Leaf RWC was decreased (p≤0.001) under salt
maize varieties. Their reduction rate was more in root than
stress in both maize varieties (Fig. 1, Table 1). Pre-sowing shoot. Seed priming with riboflavin played vital role to
seed treatment with 50ppm and 75ppm riboflavin b mitigate the adverse effect of salt stress on nutrients (K +,
significantly (p≤0.001) increased RWC in both varieties, Ca+) ions (Table 2; Fig. 4).
Pearl variety showed more RWC than Sadaf (Fig.1, Table.
1). Total chlorophyll, chlorophyll a and chlorophyll b Multivariate analysis
contents significantly (p≤0.001) decreased under 70 mM
NaCl stress in both maize varieties i.e., Pearl and Sadaf Principal component analysis (PCAs): Principal
(Fig. 2; Table 1). Pre-sowing seed treatment with 50ppm component analysis were conducted for growth,
and 75ppm riboflavin b significantly (p≤0.001) increased photosynthetic pigments, biochemical of maize under salt
chlorophyll (Chl. a, Chl. b and total chlorophyll) contents. stress and Riboflavin (RF) applications. The distance
Pearl variety of maize accumulated more chlorophyll b between eigenvectors and values of positive or negative
values demonstrated the effect of applied applications. The
(p≤0.001) contents than variety Sadaf (Fig. 2, Table 1).
PCAs for these traits showed a cumulative variability of
87.5%. Both varieties exhibited strong association by
Hydrogen peroxide (H2O 2), malondialdehyde (MDA), overlapping the eclipses. As a result of a higher saline level
anthocyanin, flavonoid and total phenolic contents: In the of 70 mM and RF supplementation (T3-75ppm), the organic
current study, H2O2, MDA, flavonoid and total phenolic osmolytes TSS, TSP, and Pro were significantly enhanced
contents were increased (p≤0.001) under salt stress in both and strongly associated with each other by loading to the
the varieties (Fig. 2; Table 1). These Riboflavin treated PCA1 side and showed higher positive eigenvalues. Both T3
plants showed decreased (p≤0.001) H2O2, MDA contents, and 70 mM NaCl were strongly interlinked with each other.
while flavonoid and total phenolic contents (p≤0.001) were The GPX and Flavo corresponded with higher positive
increased in both of the varieties under salt stress (Fig. 2; eigenvalues. The Ribo (T2) exhibited strong relation with
Table 1). Where as more accumulation was observed in MDA contents under influence of 70 mM NaCl treatments.
However, the SFW and RDW were associated with the T1
Sadaf than of Pearl because this accumulation was the
and 0 mM NaCl levels (Fig. 5a).
indication of stress exposure. RB application reduced this
accumulation rate gradually to minimize stress effect on Pearson correlation matrix: The Pearson correlation
plant (Fig. 2, Table 1). Anthocyanin contents did not change matrix showed a significant (p≤0.05) correlation for
significantly under salt or riboflavin treatment) in both maize growth, photosynthesis and biochemical traits of maize
varieties (Fig. 2, Table 1). Total phenolics and flavonoids plants under different NaCl and riboflavin treatment (Fig.
increased (p≤0.001) under salt stress in both the varieties. RF 5b). The photosynthetic pigments (Chl a, b and T. Chl)
treated plants showed increased phenolic and flavonoid were strongly and positively associated with the plant
contents in both maize varieties. However, Pearl showed biomass (SFW, RFW, SDW, RDW) and growth traits such
higher accumulation of total phenolic and flavonoid contents as LA, RL and Sl. The organic osmolytes (Prol, TSS, TSP)
than variety of Sadaf (Figs. 2 & 6; Table 1). were strongly correlated with the anthocyanin and weakly
associated with the antioxidant enzymes (SOD, POD).
Free proline, total proteins and total soluble sugars: Photosynthetic and growth traits were negatively
correlated with the MDA and H2O2 contents.
Proline accumulation was high under salt stress (p≤0.001) in
both varieties. Total proteins and total soluble sugars were Clustered heatmap: A cluster heatmap was constructed to
decreased (p≤0.001) under NaCl stress, however, riboflavin demonstrate the influential response of traits and varieties
treated plants showed increased accumulation of free under salt and riboflavin treatments (Fig. 6). The growth
proline, total proteins and total soluble sugars (p≤0.001) in traits (RL, SDW, SL and RDW) were strongly correlated
both the varieties. However, Pearl variety was higher in free with T3 and S2 of both varieties. The MDA and H2O2
proline, total proteins and total soluble sugars under both contents were negatively associated with photosynthetic
control and 70 mM NaCl stress (Table 1; Figs. 3 & 6). and growth traits with higher negative values.
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1213

Table 1. Mean square values of riboflavin induced modulation in growth, physiological and biochemical
traits of maize (Zea mays L.) under salt stress.
SOV Varieties NaCl RF V×NaCl V×RF NaCl×RF V×S×RF Error
RL 327.00*** 207.8*** 497.5*** 0.562ns 68.430* 8.763ns 0.541ns 8.60
SL 2523.4*** 636.7*** 3576.2*** 364.81*** 74.57ns 184.97** 44.28ns 13.56
RFW 83.11*** 32.30*** 160.17*** 0.0802ns 0.802 ns 0.295ns 1.082ns 0.732
SFW 1242.5 *** 2730.0*** 727.86*** 5.062ns 21.00*** 79.53*** 9.301 ns 2.968
RDW 13.08*** 137.3*** 18.370*** 0.667* 0.2205ns 0.2ns 0.004ns 0.136
SDW 11.33*** 13.44*** 36.015*** 0.071 ns 1.160ns 4.6838** 0.037 ns 0.413
LA 347.9*** 194.9*** 62.80*** 10.69** 2.310 ns 1.33147ns 5.210* 305.1
RWC 1591.1*** 449.02** 165.301* 56.52 ns 14.58ns 5.8790 ns 6.456 ns 44.51
Chl. a 0.162*** 0.484*** 0.005*** 0.000 ns 0.000ns 0.000ns 0.0006ns 0.000
Chl. b 0.069*** 0.018*** 0.0033*** 0.001*** 0.000*** 0.000*** 0.0005** 0.000
T. Chl 0.444*** 0.126*** 0.0171*** 0.0014* 0.000ns 0.0012* 0.002*** 0.0002
MDA 15.24ns 759.7*** 202.829* 2.911ns 2.927ns 34.514ns 8.311ns 51.94
H 2O 2 1.7463*** 13.09*** 21.23*** 0.194ns 1.053*** 0.2618* 0.543*** 0.074
Antho 0.0207ns 1.695ns 0.5691ns 0.000ns 0.137ns 7.5453** 1.256 ns 1.281
Flavo. 0.1514*** 1.124*** 0.0028*** 0.000ns 0.000ns 0.0000ns 0.0002ns 0.000
Phen 10.696*** 26.58*** 6.7551*** 0.052 ns 0.608 ns 0.0142ns 0.0730 ns 0.039
Prol 35.979* 57.49** 92.46*** 0.342ns 5.546ns 3.9849ns 1.4137 ns 4.826
TSP 135.6*** 9.428*** 2.677*** 0.5161* 0.354* 0.0487ns 0.3023* 0.0862
TSS 5474.6*** 1888.1*** 226.51*** 77.438*** 59.75*** 0.440ns 1.0859ns 0.5823
SOD 0.3434ns 4.7930*** 1.0687*** 0.256 ns 0.001 ns 0.0044 ns 0.0721ns 0.0807
POD 483.01ns 2042.5*** 1312.4*** 138.01*** 0.230ns 72.1630* 12.32ns 120.32
CAT 469.5*** 2070.5*** 1333.9*** 113.10* 0.1236ns 79.801* 7.6705ns 23.26
APX 0.1036* 0.8453*** 0.090* 0.000ns 0.013ns 0.0055 ns 0.0027 ns 0.0178
GPX 26.310*** 222.4*** 12.65*** 1.5365 ns 4.157 * 1.734 ns 1.258ns 1.168
Df. 1 2 2 1 2 2 2 24
*, ** and *** = Significant at 0.05, 0.01 and 0.001 levels respectively; ns = Non-significant; df = Degree of freedom.
Abbreviations: RL - Root length, SL - Shoot length, RFW - Root fresh weight, RDW - Root dry weight, SFW - Shoot fresh weight, SDW -
Shoot dry weight, LA - Leaf area, RWC- Relative water contents, Chl. - Chlorophyll, T. Chl. - Total Chlorophyll, MDA - Melondialdehyde,
Antho - Anyhocyanin, Flavo. - Flavonoid, Phenol. - Phenolics, Pro. - Proline, T.Pro. - Total proteins, TSS - Total soluble sugars, SOD -
Superoxidase dismutase, POD - Peroxidase, Cat - Catalase, APX - Ascorbate peroxidase, GPX- Guaicol peroxidase, RF - Riboflavin

Table 2. Mean square values of riboflavin induced modulation in ion contents of maize (Zea mays L.)
under salt stress.
NaCl
SOV Varieties RF V × NaCl V × RF NaCl × RF V × S × RF Error
(stress)
Shoot Na+ 658.7*** 44.44*** 100.0*** 1.777ns 17.36** 1.361ns 1.361ns 2.055
Root Na+ 744.6*** 46.6*** 111.4*** 2.25ns 23.69*** 0.861ns 1.75ns 1.833
-
Shoot Cl 73.25*** 3185.25*** 119.75*** 15.069* 0.583ns 1.083ns 2.027ns 3.277
Root Cl- 61.36*** 2826.7*** 186.86*** 34.027*** 2.1945** 13.36ns 2.027ns 2.334
2+
Shoot Ca 9.0** 560.11*** 72.86*** 0.000*** 3.583ns 0.194ns 2.583ns 1.111
2+
Root Ca 18.77*** 544.44*** 72.69*** 0.111ns 3.6944* 1.861ns 0.8611ns 1.055
Shoot K+ 1708.45*** 53.77*** 48.69*** 1.778ns 3.027ns 0.361ns 0.3611ns 1.583
Root K+ 1808.4*** 54.77*** 49.094*** 1.787ns 2.927ns 0.359ns 0.3591ns 1.493
df 1 2 2 1 2 2 2 24
*, ** and *** = Significant at 0.05, 0.01 and 0.001 levels respectively; ns = Non-significant; df = Degree of freedom.
Abbreviations: RL - Root length, SL - Shoot length, RFW - Root fresh weight, RDW - Root dry weight, SFW - Shoot fresh weight, SDW -
Shoot dry weight, LA - Leaf area, RWC- Relative water contents, Chl. - Chlorophyll, T. Chl. - Total Chlorophyll, MDA - Melondialdehyde,
Antho - Anyhocyanin, Flavo. - Flavonoid, Phenol. - Phenolics, Pro. - Proline, T.Pro. - Total proteins, TSS - Total soluble sugars, SOD -
Superoxidase dismutase, POD - Peroxidase, Cat - Catalase, APX - Ascorbate peroxidase, GPX- Guaicol peroxidase, RF - Riboflavin
1214 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

0 ppm 50 ppm 75 ppm 0 ppm 50 ppm 75 ppm

45 LSD=8.5595822 160 LSD=10.748136
40 140

Shoot length (cm)

35
Root length (cm)

120
30
100
25
80
20
15 60

10 40

5 20
0 0
LSD=2.49723197 LSD=5.0284634
25 80

70
Root fresh wt. (g plant-1)

Shoot fresh wt. (g plant-1)

20
60

15 50

40
10 30

20
5
10

0 0
LSD=0.34038687 25 LSD=1.87758060
12
Shoot dry wt. (g plant-1)
Root dry wt. (g plant-1)

10 20

8
15

6
10
4

5
2

0 0
LSD=50.9862319 LSD=19.4734051
450 60
400
Relative water contant %
Leaf area (cm2)

350 50
300 40
250
30
200
150 20
100
10
50
0 0
Control (0mM Salt stress Control (0mM Salt stress Control (0mM Salt stress Control (0mM Salt stress
NaCl) (70mM NaCl) NaCl) (70mM NaCl) NaCl) (70mM NaCl) NaCl) (70mM NaCl)
Pearl Sadaf Pearl Sadaf

Fig. 1. Morpho-physiological attributes of maize (Zea mays) plants seeds of which were pre-treated with different riboflavin levels.
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1215

0 ppm 50 ppm 75 ppm 0 ppm 50 ppm 75 ppm

LSD=0.01350237 LSD=0.00738402
0.4 0.3

0.35 0.25
Chl a (mg g-1 f.wt.)

Chl b (mg g-1 f.wt.)

0.3
0.2
0.25
0.2 0.15

0.15
0.1
0.1
0.05
0.05
0 0
LSD=0.04742478 LSD=0.79399762
0.7 9
8
0.6

H2O2 (µmol g-1 f. wt.)

7
0.5
6
T. Chl

0.4 5

0.3 4
3
0.2
2
0.1 1

0 0
LSD=21.0364018 LSD=3.30392708
40 6
MDA (nmol ml-1g-1 f. wt.)

35
5
Anthocyanin (A535)

30
4
25

20 3

15
2
10
1
5

0 0
LSD=0.02655941 LSD=0.57965795
0.9 5
Flavonoids (mg g-1 f. wt.)

Phenolic (mg g-1 f. wt.)

0.8 4.5
0.7 4
3.5
0.6
3
0.5
2.5
0.4
2
0.3 1.5
0.2 1
0.1 0.5
0 0
Control (0mM Salt stress Control (0mM Salt stress Control (0 mM Salt stress (70 Control (0 mM Salt stress (70
NaCl) (70mM NaCl) NaCl) (70mM NaCl) NaCl) mM NaCl) NaCl) mM NaCl)
Pearl Sadaf Pearl Sadaf

Fig. 2. Biochemical attributes of maize (Zea mays) plants seeds of which were pre-treated with different riboflavin levels.
1216 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

0 ppm 50 ppm 75 ppm 0 ppm 50 ppm 75 ppm

LSD=6.41243684 LSD=2.22805473
30 10

Total soluble proteins (mg g-1 f. wt.)

9
Proline (μmol g-1 f. wt.)

25 8
7
20
6
15 5
4
10
3
2
5
1
0 0
LSD=2.22805473 LSD=2.62285699
90 6
Total soluble sugar (mg g-1 f.wt)

SOD ( Units mg-1 protein)

80
5
70
60 4
50
3
40
30 2
20
1
10
0 0
LSD=32.0166484 LSD=14.0769381
1 90
CAT ( Units mg-1 protein)

0.9 80
POD (µg-1 protein)

0.8 70
0.7 60
0.6
50
0.5
40
0.4
30
0.3
20
0.2
0.1 10

0 0
LSD=0.38941560 LSD=3.15445813
1.4 20
1.2 18
GPX (µg-1 protein)
Apx (µg-1 protein)

16
1 14
0.8 12
10
0.6 8
0.4 6
4
0.2 2
0 0
Control (0 mM Salt stress (70 Control (0 mM Salt stress (70 Control (0 mM Salt stress (70 Control (0 mM Salt stress (70
NaCl) mM NaCl) NaCl) mM NaCl) NaCl) mM NaCl) NaCl) mM NaCl)

Pearl Sadaf Pearl Sadaf

Fig. 3. Organic osmolytes and antioxidant enzyms activities of maize (Zea mays L.) plants seeds of which were pre-treated with different riboflavin levels.
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1217

0ppm 50ppm 75ppm 0ppm 50ppm 75ppm

LSD = 4.1790760 LSD= 3.94847044
35 45
40
Shoot Na+ (mg/g DW)

Root Na+ (mg/g DW)

30
35
25
30
20 25

15 20
15
10
10
5 5
0 0
LSD = 5.278095 LSD= 4.4591
45 40

40 35
Shoot Cl- (mg/g DW)

35
Root Cl- (mg/g DW)
30
30
25
25
20
20
15
15
10
10
5 5

0 0
LSD = 3.0779087 LSD = 2.9908731
20 18
18 16
Shoot Ca+ (mg/g DW)

Root Ca+ (mg/g DW)

16 14
14 12
12
10
10
8
8
6
6
4 4

2 2
0 0
LSD = 3.668866 LSD = 3.57477
30 30
Shoot K+ (mg/g DW)

Root K+ (mg/g DW)

25 25

20 20

15 15

10 10

5 5

0 0
Control (0 mM Salt stress (70 Control (0 mM Salt stress (70 Control (0 mM Salt stress (70 Control (0 mM Salt stress (70
NaCl) mM NaCl) NaCl) mM NaCl) NaCl) mM NaCl) NaCl) mM NaCl)
Pearl Sadaf Pearl Sadaf

Fig. 4. Mineral ion contents of maize (Zea mays L.) plants seeds of which were pre-treated with different riboflavin levels.
1218 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

T3
70 mM
70 mM T2
T3
70 mM 70 mM
T2
T3 70 mM
0 mM T1
0 mM T2 0 mM
0 mM T1
0 mM
0 mM

Fig. 5. a) PCA biplot, b) Pearson correlation 4, a) 441(T1- 0 ppm, T2-50 ppm, T3-75 ppm).
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1219

Fig. 6. Cluster heatmap for growth, photosynthetic pigments, biochemical of maize under Salt stress (S1-0 mM & S2-70 mM NaCl),
Riboflavin (T1-0 ppm, T2-50 ppm, T3-75 ppm) and varieties (P-pearl, S, Sadaf).

Discussion cell, imbalanced supply of nutrients and damages to

membrane integrity (Akhtar et al., 2015, Sobhanian et al.,
Salt stress is ubiquitously threatening factor for crop 2010). Vitamins play important role to enhance plant
growth, development and yield. Salt containing soils growth rate and biomass by reducing reactive oxygen
consist of large amount of soluble salts and exchangeable species (ROS) production and enabling plants to uptake
sodium ions in root area. Sodium chloride stress restricts essential nutrients (Kaya et al., 2015). Similar results were
the growth rate of commercial crops; as it harms more than defined by Safdar et al., (2019).
800 million ha of land worldwide (Anon., 2000). Salt stress Photosynthesis is important food producing process
reduces plant growth as it provokes oxidative stress, ion in plants. Plants subjected to salinity stress show reduced
imbalance and many secondary stresses like oxidative photosynthetic rate. Present study depicted reduced
stress and lack of nutrients (Deinlein et al., 2014). In this photosynthetic pigments (chl. a chl. b and total
experiment, two maize cultivars were examined to measure chlorophyll) after plants subjected to salinity stress.
the morpho-physiological responses against saline soil (salt However, riboflavin application played a positive role to
stress) and mitigating effect of vitamin B2. A significant enhance these photosynthetic pigments (Fig. 2, Table. 1).
reduction in the plant growth and dry weight along with Chlorophyll reduction may be due to increased
leaf area was noticed with exposure of salt stress to these cholorophylase, a chlorophyll degradation enzyme
maize cultivars. But riboflavin (RF) priming showed produced as a result of elevated salinity level (Noreen &
enhanced plant growth rate and biomass (Fig. 1, Table. 1). Ashraf, 2009). Another reason might be stomatal closure
The mechanism behind this growth, leaf area and relative due to water deficiency by increased nutrient uptake in
water content (RWC) is low amount of water uptaking by presence of high salt level (Chatrath et al., 2000). One
plant which causes slow plant growth. Another reason more reason for reduced photosynthetic rate might be
might be entrance of salt in the plant cell and cause cell decreased efficiency of PS-II and reduced photon yield
injury and cell death (Munns, 2005). The limited supply of under salinity stress (Yang & Lu, 2005; Chu-Um &
water, aeration and nutrients due to salt stress leads to Kirdmanee, 2009). Vitamins showed positive response in
decline in plant biomass (Attia et al., 2008). Another reason increasing chlorophyll contents by increasing stomatal
of growth suppression under salt stress is the shrinkage of opening time and supressing production of chlorophyll
1220 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

degradation enzyme in salt subjected plants (Wahid & which is elevated under salinity stress resulting in
Jamil, 2009). Same findings were observed in pumpkin osmotic potential regulation. This is key amino acid in
(Sevengor et al., 2011). scavenging free radicals (Ashraf & Harris, 2004).
Malondialdehyde (MDA) is the indicator of stress Present experiment showed that free proline
exposure, which results in membrane impairment when accumulation under salt stress and riboflavin (vitamin)
plant is exposed to salt stress (Katsuhara et al., 2005). In application improved its accumulation rate (Fig. 3, Table
this experiment, MDA and hydrogen peroxide were 1). Plants facing high salinity stress accumulate more
accumulated under salinity stress and RF application proline contents in order to resist abiotic stresses and to
decreased this accumulation rate (Fig. 2; Table 1). produce plant tolerance against these environmental
Membrane breakdown, ion leaking, lipid peroxidation and stresses. Proline accumulation might be due to osmotic
difficulty in nutrient uptake may be the reason of adjustment, and maintaining plant cell structure under
accumulated MDA (as membrane damage is associated salinity stress (Turan et al., 2009). Another possible
with MDA accumulation) and hydrogen peroxides in plants reason of increased free proline accumulation might be
to compete with increased production of oxidative stress the upregulation of pyroline-5-carboxylate and down
indicators (Sacała, 2017). Vitamins decrease ion leakage regulation of PDH (proline dehydrogenase enzyme).
and membrane breakdown because they act as growth Proline plays an important role in radicle detoxification
regulator. Exogenously applied vitamin to seeds showed and enzyme protection (Ashraf & Foolad, 2007).
reduced accumulation of MDA and hydrogen peroxide Increaesd proline accumulation under salinity stress was
under salinity stress (Tunc-Ozdemir et al., 2009). Khan et also observed in tomato (Amini & Ehsanpour, 2005) and
al., (2002) also concluded that MDA and H2O2 are in wheat (Turan et al., 2007). Application of vitamin (vit.
accumulated in rice on salinity stress exposure. Same B2) showed positive improvement in proline content.
findings were observed in sorghum (Huang, 2018). Tuna et al., (2013) studied the effects of vitamins (vit.
Anthocyanin and flavonoids are water soluble B2) on proline accumulation in maize under salinity
pigments the accumulation rate of which varied in plants stress. Similar findings were observed in sunflower
exposed to abiotic stresses. Anthocyanin accumulation (Sayed & Gadallah, 2002).
decreased, while flavonoids accumulation was increased Soluble proteins are the stress indicators in plants. In the
in vegetative tissues of plants that were subjected to present study, total soluble proteins showed an increase
salinity stress. The current study showed that anthocyanin under salt stress in both maize cultivars, more accumulation
were accumulated under non stress conditions, while was observed in salt tolerant variety than sensitive one. It has
flavonoids were accumulated under salt stress conditions. been described that after vitamin application plants species
RF application increased their accumulation rate (Fig. 2, accumulate greater contents of protein under salinity stress
Table. 1). Both anthocyanin and flavonoids play crucial (Fig. 3, Table 1). This protein accumulation might be due to
role towards oxidative stress (Pervaiz et al., 2017) under synthesis of osmotin like protein which are involved in cell
salinity stress. When vitamins are applied exogenously, wall modification under abiotic stress condition to enhance
they show positive response to compete stress in order to osmotic adjustment and plant survival rate (Abdel Latef,
produce increased anthocyanin and flavonoid 2010). Same results were concluded under salinity stress in
concentration (Bahmani et al., 2015). When a plant is wheat and in maize (Ali et al., 2022).
exposed to abiotic stress, reactive oxygen species are Data of current study showed increased soluble
generated as a stress signal and ultimately activation of sugars accumulation under salt stress, however, maize
flavonoid and anthocyanin is regulated. This differential plants raised from seeds treated with RF showed further
accumulation is due to increased oxidative stress and accumulation in order to combat with salt stress
enhanced ROS which result in reduced osmotic damage, conditions (Fig. 3; Table 1). This increase in total
photo-protection and quenching of ROS. soluble sugars might be due to increased osmotic
Plants are adaptive to face many abiotic stresses i.e., potential and increased water absorption after salinity
salinity stress (Zhoa et al., 2020). This ability is increased induction (Abdelgawad et al., 2016, Nemati et al.,
due to metabolites (phenolics) in plants (Ali et al., 2006). 2011). It is an important feature of any plant to
Phenolics are important non enzymatic antioxidants having accumulate sugars under salinity exposure (Ashraf &
ability to donate hydrogen ions and ultimately accumulated Harris, 2004). Vitamins play antioxidant role in plants
under salinity stress (Posmyk et al., 2009). In this study, to mitigate deleterious effect of salinity stress. Soluble
increased total phenolic content was observed in both Z. sugars are increased due to vitamin priming thus
mays cultivars. Under salt stress Vitamin application strengthening osmotic potential (Sayed & Gadallah,
showed positive accumulation of phenolic concentration 2002). Similiar findings were reported in tomato (Shibli
under stress conditions (Fig. 3, Table 1). Phenolics have et al., 2007; Turan et al., 2007) and in Brassica napus
ability of ROS scavenging and hinders the conversion of (Ahmadi et al., 2018).
H2O2 to free radicals under salinity stress (Pearse et al., Abiotic stresses produce reactive oxygen species in
2005). Phenolics protect plasma membrane by reducing the plants resulting in membrane damaging, deoxyribose
oxidative stress effect of ROS and increasing the nucleic acid damage, loss of carbohydrates and lipids
production rate of antioxidants. Navarro et al., (2006) ultimately result in oxidative stress. Present study
conducted similar studies on pepper plant. showed increased antioxidant enzymes (POD, SOD,
Prolines are water soluble compatible solutes. These CAT, APX and GPX) activities under sodium chloride
are member of amino acid group the accumulation of stress and riboflavin pre-treatment showed more
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1221

increase in the activities of these antioxidant enzymes Abdel Latef, A. 2010. Changes of antioxidative enzymes in
(Fig. 3; Table 1). Antioxidant enzymes (SOD, POD, salinity tolerance among different wheat cultivars. Cereal
CAT, APX and GPX) are able to scavenge deleterious Res. Comm., 38(1): 43-55.
effect of ROS, where POD has hydrogen peroxide Abdelgawad, H., G, Zinta, M.M. Hegab, R. Pandey, H. Asard and
W. Abuelsoud. 2016. High salinity induces different
scavenging ability and SOD has singlet oxygen oxidative stress and antioxidant responses in maize seedlings
scavenging ability. Antioxidant enzymes i.e., CAT and organs. Front. Plant Sci., 7: 276.
APX with their high activity rate decrease the hydrogen Abdulhamed, Z.A., A.O. Alfalahi and N.M. Abood. 2020.
peroxide level in plant cells resulting in improved December. Riboflavin and Cultivars affecting genetic
stability of membrane and carbon dioxide fixation, parameters in maize (Zea mays L.). In: AIP Conference
because many chloroplast consisting of Calvin cycle proceedings (Vol. 2290, No. 1, p. 020020). AIP Publishing
enzymes are sensitive to H 2O2 (restrict carbon dioxide LLC.
fixation) (Yamazaki et al., 2003; Esfandiari et al.,2007). Ahmad, M., Q. Ali, M. M. Hafeez and A. Malik. 2021. Improvement
Increased antoxidative activities of enzymes are closely for biotic and abiotic stress tolerance in crop plants. J. Biol. Res.,
2021: 1 https://doi.org/10.54112/bcsrj.v2021i1.50
related to decreased oxidative stress (Candan & Tarhan, Ahmadi, F.I., K. Karimi and P.C. Struik. 2018. Effect of
2003). A high level of H2O2 directly inhibits CO 2 exogenous application of methyl jasmonate on physiological
fixation. Antioxidant enzymes showed increased and biochemical characteristics of Brassica napus L. cv.
activities after vitamin (vitamin B2) treatment to seeds Talaye under salinity stress. S. Afr. J. Bot., 115: 5-11.
(Chi et al., 2021). Because vitamins act as coenzymes in Ahn, I.P., S. Kim and Y.H. Lee. 2005. Vitamin B1 functions as an
metabolic pathways (Goyer et al., 2010) to protect the activator of plant disease resistance. Plant Physiol., 138(3):
plant from abiotic stress by increasing tolerance level of 1505-1515.
plants against oxidative stress (Ahn et al., 2005). Similar Akhtar, S.S., M.N. Andersen, M. Naveed, Z.A. Zahir and F. Liu.
results were also recorded in potato (Sattar et al., 2021) 2015. Interactive effect of biochar and plant growth-
promoting bacterial endophytes on ameliorating salinity
and tobacco (Wang et al., 2010). stress in maize. Funct. Plant Biol., 42(8): 770-781.
This excessive increase in Na+ and Cl- ions results in Alayafi, A.A.M. 2020. Exogenous ascorbic acid induces systemic
nutrients imbalance, effects osmotic regulation which heat stress tolerance in tomato seedlings: Transcriptional
ultimately results in ion toxicity (Katerji et al., 2004; regulation mechanism. Environ. Sci. Pollut. Res., 27(16):
Arzani, 2008). Uptaking mechanism of ions is disturbed in 19186-19199.
membranes which results in increased Cl- translocation in Ali, B., X. Wang, M.H. Saleem, M.A. Azeem, M.S. Afridi, M.
shoots (Yousif et al., 1972; Yong et al., 2020). In this study, Nadeem, M. Ghazal, T. Batool, A. Qayuum, A. Alatawi and
salt stress increased the sodium and chloride ion S. Ali. 2022. Bacillus mycoides PM35 reinforces
concentration in roots and shoots which ultimately photosynthetic efficiency, antioxidant defense, expression of
stress-responsive genes, and ameliorates the effects of
disturbed growth rate and proper functioning of plant. salinity stress in maize. Life, 12(2): 219.
Same results were observed by Chavan & Karadge (1986) Ali, M.B., S. Khatun, E.J. Hahn and K.Y. Paek. 2006.
and Turan et al., (2007). With the increase in salinity level, Enhancement of phenylpropanoid enzymes and lignin in
potassium is decreased in root and shoot because plasma Phalaenopsis orchid and their influence on plant
membrane is depolarized by sodium ions and results in acclimatisation at different levels of photosynthetic photon
potassium ion leakage (Cramer et al., 1985). In current flux. Plant Growth Regul., 49(2): 137-146.
study, potassium ions decreased under salt stress. Similar, Amini, F. and A.A. Ehsanpour. 2005. Soluble proteins, proline,
results were observed by Karmoker et al., (2008). carbohydrates and Na+/K+ changes in two tomato
(Lycopersicon esculentum Mill.) cultivars under in vitro salt
stress. Amer. J. Biochem. Biotechnol., 1(4): 204-208.
Conclusion Anonymous. 2000. Global network on integrated soil management
for sustainable use of salt-affected soils. Available in:
Present study concluded that salt stress exerted drastic http://www.fao.org/ag/AGL/agll/spush/intro.htm (28 Jan.2015).
effect on growth of maize plants by increasing reactive Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts.
oxygen species concentration. Pre-sowing seed treatment Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24(1): 1.
with riboflavin showed positive response in improving salt Arzani, A. 2008. Improving salinity tolerance in crop plants:
stress tolerance in maize cultivars (Pearl and Sadaf). Of all a biotechnological view. Vitro Cell. Dev. Biol. Plant, 44:
the studied attributes including shoot and root fresh and dry 373-383.
weight and shoot and root length, chlorophyll contents, Asada, K. 1992. Ascorbate peroxidase–a hydrogen peroxide‐
scavenging enzyme in plants. Physiol. Plant., 85(2): 235-241.
flavonoids, total phenolics, total soluble sugars, total
Ashraf, M. and M.R. Foolad. 2007. Roles of glycine betaine and
soluble proteins, free proline and activities of catalase, proline in improving plant abiotic stress resistance. Environ.
ascorbate peroxidase and guaiacol peroxidase and mineral Exp. Bot., 59(2): 206-216.
ions (K+ and Ca2+) Pearl variety of maize than that of Sadaf. Ashraf, M. and P.J.C. Harris. 2004. Potential biochemical
The results obtained showed were higher in variety Pearl indicators of salinity tolerance in plants. Plant Sci., 66(1):
was more tolerant to salt stress compared to Sadaf. 3-16.
Attia, H., N. Arnaud, N. Karray and M. Lachaâl. 2008. Long‐term
References effects of mild salt stress on growth, ion accumulation and
superoxide dismutase expression of Arabidopsis rosette
Abd El-Samad, H.M., D. Mostafa and K.N. Abd El-Hakeem. leaves. Physiol. Plant, 132(3): 293-305.
2017. The combined action strategy of two stressses, salinity Bahmani, K., S.A.S. Noori, A.I. Darbandi and A. Akbari. 2015.
and Cu++ on growth, metabolites and protein pattern of Molecular mechanisms of plant salinity tolerance: A review.
wheat plant. Amer. J. Plant Sci., 8(3): 625-643. Aust. J. Crop Sci., 9(4): 321-336.
1222 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

Bates, L.S., R.P. Waldren and I.D. Teare. 1973. Rapid Hadia, E., A. Slama, L. Romdhane, M. Cheikh, M.A.S. Fahej and
determination of free proline for water-stress studies. Plant L. Radhouane. 2022. Seed priming of bread wheat varieties
Soil, 39(1): 205-207. with growth regulators and nutrients improves salt stress
Bradford, M.M. 1976. A rapid and sensitive method for the tolerance particularly for the local genotype. J. Plant Growth
quantitation of microgram quantities of protein utilizing the Regul., 1-15.
principle of protein-dye binding. Anal. Biochem., 72(1-2): Hafeez, M.B., N. Zahra, K. Zahra, A. Raza, A. Khan, K. Shaukat
248-254. and S. Khan. 2021. Brassinosteroids: Molecular and
Cakmak, I. and W.J.T. Hors. 1991. Effect of aluminium on lipid physiological responses in plant growth and abiotic stresses.
peroxidation, superoxide dismutase, catalase, and Plant Stress, 2: 100029.
peroxidase activities in root tips of soybean (Glycine max). Hasanuzzaman, M., M.H.M. Bhuyan, F. Zulfiqar, A. Raza, S.M.
Physiol. Plant, 83(3): 463-468. Mohsin and J.A. Mahmud. 2020. Reactive oxygen species
Candan, N. and L. Tarhan. 2003. The correlation between and antioxidant defense in plants under abiotic stress:
antioxidant enzyme activities and lipid peroxidation levels in Revisiting the crucial role of a universal defense regulator.
Mentha pulegium organs grown in Ca2+, Mg2+, Cu2+, Zn2+ Antioxidants, 9(8): 681.
and Mn2+ stress conditions. Plant Sci., 165(4): 769-776. Hassanein, A.M., H.M. Azooz and F.A. Faheed. 2002. Riboflavin,
Carleton, A.E. and W.H. Foote. 1965. A comparison of methods vitamin B2 treatments counteract the adverse effect of
for estimating total leaf area of barley plants 1. Crop Sci., salinity on growth and some relevant Physiologyical
5(6): 602-603. responses of Hibiscus sabdariffa L. seedlings. Bull. Fac.
Chance, B. and A.C. Maehly. 1955. Assay of catalases and Sci., Assiut. Univ. (Egypt), 31: 295-303.
peroxidases. 136: 764-775. Huang, R.D. 2018. Research progress on plant tolerance to soil
Chatrath, A., P.K. Mandal and M. Anuradha. 2000. Effect of salinity and alkalinity in sorghum. J. Integr. Agric., 17(4):
secondary salinization on photosynthesis in fodder oat (Avena 739-746.
sativa L.) genotypes. J. Agron. Crop Sci., 184(1): 13-16. Jiadkong, K., M. Nampei, S. Wangsawang and A. Ueda. 2023.
Cha-Um, S. and C. Kirdmanee. 2009. Effect of salt stress on Riboflavin seed priming activates OsNHXs expression to
proline accumulation, photosynthetic ability and growth alleviate salinity stress in rice seedlings. J. Plant Growth
characters in two maize cultivars. Pak. J. Bot., 41(1): 87-98. Regul., 42(5): 3032-3042.
Chavan, P.D. and B.A. Karadge. 1986. Growth, mineral nutrition, Johnson, C.M. and A. Ulrich. 1959. Analytical methods for
organic constituents and rate of photosynthesis in Sesbania use in plant analysis. California Agric. Exp. Station Bull.,
grandiflora L. grown under saline conditions. Plant Soil, 93: 766: 44-45.
395-404. Jones, M.M. and N.C. Turner. 1978. Osmotic adjustment in leaves
Chi, Y.X., L. Yang, C.J. Zhao, I. Muhammad, X.B. Zhou and H. of sorghum in response to water deficits. Plant Physiol.,
De Zhu. 2021. Effects of soaking seeds in exogenous 61(1): 122-126.
vitamins on active oxygen metabolism and seedling growth Julkunen-Titto, R. 1985. Phenolic constituents in the levels of
under low-temperature stress. Saudi J. Biol. Sci., 28(6): northern willows: methods for precursors of clarified apple
3254-3261. juice sediment. J. Food Sci., 33: 254-257.
Cramer, G.R., A. Lauchli and V.S. Polito. 1985. Displacement of Karadeniz, F., H.S. Burdurlu, N. Koca and Y. Soyer. 2005.
Ca2+ by Na+ from the plasmalemma of root cell. A primary Antioxidant activity of selected fruits and vegetables grown
response to salt stress? Plant Physiol., 79: 207-211. in Turkey. Turk. J. Agric. For., 29(4): 297-303.
Deinlein, U., A.B. Stephan, T. Horie, W. Lu, G. Xu and J.I. Karmoker, J.L., S. Farhana and P. Rashid. 2008. Effects of salınıty
Schroeder. 2014. Plant salt-tolerance mechanisms. Trends on ıon accumulatıon ın maıze (Zea mays L. CV. BARI-7).
Plant Sci., 19(6): 371-379. Bangladesh J. Bot., 37: 203-205.
Díaz, F.J., J.C. Sanchez-Hernandez and J.S. Notario. 2021. Katerji, N., J. W. van Hoorn, A. Hamdy and M. Mastrorilli. 2004.
Effects of irrigation management on arid soils enzyme Comparison of corn yield response to plant water stress
activities. J. Arid Environ., 185: 104330. caused by salinity and by drought. Agric. Water Manag., 65:
Dikobe, T.B., B. Mashile, R.P. Sinthumule and O. Ruzvidzo. 95-101.
2021. Distinct morpho-physiological responses of maize to
Katsuhara, M., T. Otsuka and B. Ezaki. 2005. Salt stress-induced
salinity stress. Amer. J. Plant Sci., 12(6): 946-959.
lipid peroxidation is reduced by glutathione S-transferase,
Esfandiari, E., M.R. Shakiba, S.A. Mahboob, H. Alyari and M.
Toorchi. 2007. Water stress, antioxidant enzyme activity and but this reduction of lipid peroxides is not enough for a
lipid peroxidation in wheat seedling. J. Food Agric. recovery of root growth in Arabidopsis. Plant Sci., 169(2):
Environ., 5(1): 149. 369-373.
Fu, Z., P. Wang, J. Sun, Z. Lu, H. Yang, J. Liu and T. Li. 2020. Kaya, C., M. Ashraf, M.N. Alyemeni, F.J. Corpas and P. Ahmad.
Composition, seasonal variation, and salinization 2020. Salicylic acid-induced nitric oxide enhances arsenic
characteristics of soil salinity in the Chenier Island of the toxicity tolerance in maize plants by upregulating the
Yellow River Delta. Glob. Ecol. Conserv., 24: e01318. ascorbate-glutathione cycle and glyoxalase system. J
Garg, M., A. Sharma, S. Vats, V. Tiwari, A. Kumari, V. Mishra Hazard. Mater., 399: 123020.
and M. Krishania. 2021. Vitamins in cereals: a critical Kaya, C., M. Ashraf, O. Sonmez, A.L. Tuna, T. Polat and S.
review of content, health effects, processing losses, Aydemir. 2015. Exogenous application of thiamin
bioaccessibility, fortification, and biofortification strategies promotes growth and antioxidative defense system at
for their improvement. Front. Nutr., 8: 586815. initial phases of development in salt-stressed plants of two
Giannopolitis, C.N. and S.K. Ries. 1977. Superoxide dismutases: I. maize cultivars differing in salinity tolerance. Acta.
Occurrence in higher plants. Plant Physiol., 59(2): 309-314. Physiol. Plant, 37(1): 1-12.
Gorji, T., A. Yildiri, N. Hamzehpou, A. Tanik and E. Sertel. 2020. Kazemi, K. and H. Eskandari. 2012. Does priming improve seed
Soil salinity analysis of Urmia Lake Basin using Landsat-8 performance under salt and drought stress. J. Basic Appl. Sci.
OLI and Sentinel-2A based spectral indices and electrical Res., 2(4): 3503-3507.
conductivity measurements. Ecol. Indic., 112: 106173. Khan, M.H., K.L. Singha and S.K. Panda. 2002. Changes in
Goyer, A. 2010. Thiamine in plants: aspects of its metabolism and antioxidant levels in Oryza sativa L. roots subjected to NaCl-
functions. Phytochemistry, 71(14-15): 615-1624. salinity stress. Acta. Physiol. Plant, 24(2): 145-148.
RIBOFLAVIN-INDUCED SALT STRESS TOLERANCE IN MAIZE 1223

Khatun, S., T.S. Roy, M.N. Haque and B. Alamgir. 2016. Effect Sobhanian, H., N. Motamed, F.R. Jazii, T. Nakamura and N.
of plant growth regulators and their time of application on Komatsu. 2010. Salt stress induced differential proteome
yield attributes and quality of soybean. Int. J. Plant Soil Sci., and metabolome response in the shoots of Aeluropus
11(1): 1-9. lagopoides (Poaceae), a halophyte C4 plant. J. Proteome.
Lee, D.H., Y.S. Kim and C.B. Lee. 2001. The inductive responses Res., 9(6): 2882-2897.
of the antioxidant enzymes by salt stress in the rice (Oryza Tuna, A.L., C. Kaya, H. Altunlu and M. Ashraf. 2013.
sativa L.). J. Plant Physiol., 158(6): 737-745. Mitigation effects of non-enzymatic antioxidants in maize
Munns, R., S. Goyal and J. Passioura. 2005. Salinity stress and its (‘Zea mays’ L.) Plants under salinity stress. Aust. J. Crop.
mitigation. University of California, Davis. Sci., 7: 1181-1188.
Nadarajah, K.K. 2020. ROS homeostasis in abiotic stress Tunc-Ozdemir, M., G. Miller, L. Song, J. Kim, A. Sodek, S.
tolerance in plants. Int. J. Mol. Sci., 21(15): 5208. Koussevitzky, A.N. Misra, R. Mittler and D. Shintani. 2009.
Navarro, J.M., P. Flores, C. Garrido and V. Martinez. 2006. Thiamin confers enhanced tolerance to oxidative stress in
Changes in the contents of antioxidant compounds in pepper Arabidopsis. Plant Physiol., 151(1): 421-432.
fruits at different ripening stages, as affected by salinity. Turan, M. A., V. Katkat and S. Taban. 2007. Variations in proline,
Food Chem., 96(1): 66-73. chlorophyll and mineral elements contents of wheat plants
Nemati, I., F. Moradi, S. Gholizadeh, M.A. Esmaeili and M.R. grown under salinity stress. J. Agron., 6(1):
Bihamta. 2011. The effect of salinity stress on ions and Turan, M.A., A.H.A. Elkarim, N. Taban and S. Taban. 2009.
soluble sugars distribution in leaves, leaf sheaths and roots Effect of salt stress on growth, stomatal resistance, proline
of rice (Oryza sativa L.) seedlings. Plant Soil & Environ., and chlorophyll concentrations on maize plant. Afr. J. Agric.
57(1): 26-33. Res., 4(9): 893-897.
Noreen, Z. and M. Ashraf. 2009. Changes in antioxidant enzymes Turan, M.A., N. Türkmen and N. Taban. 2007. Effect of NaCl on
and some key metabolites in some genetically diverse stomatal resistance and proline, chlorophyll, Na, Cl and K
cultivars of radish (Raphanus sativus L.). Environ. Exp. Bot., concentrations of lentil plants J. Agron., 6: 378-381.
67(2): 395-402. Vaughan, M.M., A. Block, S.A. Christensen, L.H. Allen and E.A.
Pearse, I., K.D. Heath and J.M. Cheeeseman. 2005. A partial Schmelz. 2018. The effects of climate change associated
characterization of peroxidase in Rhizophora mangle. Plant abiotic stresses on maize phytochemical defenses.
Cell Environ., 28: 612-622. Phytochem. Rev., 17(1): 37-49.
Pervaiz, T., J. Songtao, F. Faghihi, M.S. Haider and J. Fang. 2017. Velikova, V., I. Yordanov and A. Edreva. 2000. Oxidative stress
Naturally occurring anthocyanin, structure, functions and and some antioxidant systems in acid rain-treated bean
biosynthetic pathway in fruit plants. J. Plant Biochem. plants: protective role of exogenous polyamines. Plant Sci.,
Physiol., 5(2): 1-9. 151(1): 59-66.
Posmyk, M.M., R. Kontek and K.M. Janas. 2009. Antioxidant Waheed AL-Mayahi, A.M. 2016. Influence of salicylic acid (SA)
enzymes activity and phenolic compounds content in red and ascorbic acid (ASA) on 'In vitro' propagation and salt
cabbage seedlings exposed to copper stress. Ecotoxicol. tolerance of date palm ('Phoenix dactylifera'L.) cv.'Nersy'.
Environ. Saf., 72(2): 596-602. Aust. J. Crop Sci., 10(7): 969-976.
Sacała, E. 2017. The influence of increasing doses of silicon on Wahid, A. and A. Jamil. 2009. Inducing salt tolerance in canola
maize seedlings grown under salt stress. J. Plant Nutr., (Brassica napus L.) by exogenous application of
40(6): 819-827. glycinebetaine and proline: response at the initial growth
Safdar, H., A. Amin, Y. Shafiq, A. Ali, R. Yasin, A. Shoukat, M.U. stages. Pak. J. Bot., 41(3): 1311-1319.
Hassan and M.I. Sarwar. 2019. A review: Impact of salinity Wang, Y., C. Gao, Y. Liang, C. Wang, C. Yang and G. Liu. 2010.
on plant growth. Nat. Sci., 17(1): 34-40. A novel bZIP gene from Tamarix hispida mediates
Santpoort, R. 2020. The drivers of maize area expansion in Sub- physiological responses to salt stress in tobacco plants. J.
Saharan Africa. How policies to boost maize production Plant Physiol., 167(3): 222-230.
overlook the interests of smallholder farmers. Land., 9(3): 68. Waqas, M.A., X. Wang, S.A. Zafar, M.A. Noor, H.A. Hussain,
Sattar, F.A., B.T. Hamooh, G. Wellman, M. Ali, S.H. Shah, Y. M.A. Nawaz and M. Farooq. 2021. Thermal stresses in maize:
Anwar and M.A.A. Mousa. 2021. Growth and biochemical Effects and management strategies. Plants, 10(2): 293.
responses of potato cultivars under In vitro lithium chloride Wei, Y., J. Ding, S. Yang, F. Wang and C. Wang. 2021. Soil
and mannitol simulated salinity and drought stress. Plants, salinity prediction based on scale-dependent relationships
10(5): 924. with environmental variables by discrete wavelet transform
Sayed, S.A. and M.A.A. Gadallah. 2002. Effects of shoot and root in the Tarim Basin. Catena, 196: 104939.
application of thiamin on salt-stressed sunflower plants. Wichelns, D. and M. Qadir. 2015. Achieving sustainable
Plant Growth Regul., 36(1): 71-80. irrigation requires effective management of salts, soil
Sevengor, S., F. Yasar, S. Kusvuran and S. Ellialtioglu. 2011. The salinity, and shallow groundwater. Agric. Water Manag.,
effect of the effect of salt stress on growth, chlorophyll 157: 31-38.
content, lipid peroxidation and antioxidative enzymes of Yamazaki, J.Y., A. Ohashi, Y. Hashimoto, E. Negish, S. Kumagai,
pumpkin seedling. Afr. J. Agric. Res., 6(21): 4920-4924. T. Kubo, T. Oikawa, E. Maruta and Y. Kamimura. 2003.
Shiade, S.R.G. and B. Boelt. 2020. Seed germination and seedling Effects of high light and low temperature during harsh winter
growth parameters in nine tall fescue varieties under salinity on needle photodamage of Abies mariesii growing at the
stress. Acta Agri. Scand., 70(6): 485-494. forest limit on Mt. Norikura in Central Japan. Plant Sci.,
Shibli, R.A.M., G.G. Kushad, Yousef and M.A. Lila. 2007. 165(1): 257-264.
Physiological and biochemical responses of tomato Yang, X. and C. Lu. 2005. Photosynthesis is improved by
microshoots to induce salinity stress with associated exogenous glycinebetaine in salt‐stressed maize plants.
ethylene accumulation. J. Plant Growth Regul., 51: 159-169. Physiol. Plant, 124(3): 343-352.
Singh, A. 2022. Soil salinity: A global threat to sustainable Yemm, E.W. and A. Willis. 1954. The estimation of carbohydrates
development. Soil Use Manag., 38(1): 39-67. in plant extracts by anthrone. Biochem. J., 57(3): 508.
1224 NOSHEEN IFTIKHAR & SHAGUFTA PERVEEN

Yong, M.T., C.A. Solis, B. Rabbi, S. Huda, R. Liu, M. Zhou, L. Zahra, N., Z. Raza and S. Mahmood. 2020. Effect of salinity
Shabala, G. Venkatraman, S. Shabala and Z. Chen 2020. stress on various growth and physiological attributes of two
Leaf mesophyll K+ and Cl− fluxes and reactive oxygen contrasting maize genotypes. Braz. Arch. Biol. Tech., 63.
species production predict rice salt tolerance at reproductive Zhang, C., Y. Ma, X. Zhao and J. Mu. 2009. Influence of
stage in greenhouse and field conditions. Plant Growth copigmentation on stability of anthocyanins from purple
Regul., 92(1): 53-64. potato peel in both liquid state and solid state. J. Agric. Food
Yoshii, K., K. Hosomi, K. Sawane and J. Kunisawa. 2019. Chem., 57: 9503-9508.
Zhao, C., H. Zhang, C. Song, J.K. Zhu and S. Shabala. 2020.
Metabolism of dietary and microbial vitamin B family in the
Mechanisms of plant responses and adaptation to soil
regulation of host immunity. Front. Nutr., 6: 48.
salinity. The Innovation, 1(1): 100017.
Yousif, H.Y., F.T. Bingham and D.M. Yermason. 1972. Growth, Zhou, T., H. Li, M. Shang, D. Sun, C. Liu and G. Che. 2021 Recent
mineral composition, and seed oil of sesame (Sesamum analytical methodologies and analytical trends for riboflavin
indicum L.) as affected by NaCl. Soil Sci. Soc. Amer. Proc., (vitamin B2) analysis in food, biological and pharmaceutical
36: 450-453. samples. TrAC Trends Anal. Chem., 143: 116412.

(Received for publication 16 November 2022)