Braz. J.
Bot
DOI 10.1007/s40415-015-0174-6
Modulations in plant water relations and tissue-specific
osmoregulation by foliar-applied ascorbic acid and the induction
of salt tolerance in maize plants
Sidra Jamil1 • Qasim Ali1 • Muhammad Iqbal1 • Muhammad Tariq Javed1
Wasif Iftikhar1 • Faisal Shahzad1 • Rashida Perveen2
Received: 10 November 2014 / Accepted: 15 May 2015
 Botanical Society of Sao Paulo 2015
Abstract Plant water status and cellular osmotic adjust-
ments play a key role in the salt tolerance of plants. An
experiment was conducted to assess the influence of foliar-
applied ascorbic acid (AsA) on the plant water relations,
cellular osmotic adjustments, uptake, and accumulation of
different ions in the two high yielding maize cultivars
(Agaiti-2002 and EV-1098) at early growth stage under salt
stress (120 mM NaCl). Salt stress altered growth, water
relation attributes, osmolytes accumulation, and decreased
the uptake of K?, Ca2?, P, and N, while increased the
uptake and accumulation of Na? in different plant parts
(leaf, stem, and root). Exogenous AsA increased the ac-
cumulation of AsA in different plant parts, particularly in
leaves and roots. Moreover, AsA application resulted in the
accumulation of proline and glycinebetaine (GB) in the
roots and leaves of both maize cultivars when under salt
stress. Foliar application of AsA also increased the uptake
of minerals such as K?, Ca2?, P, and N and decreased the
uptake and accumulation of Na? in different plant parts.
Taken together, the AsA-induced enhanced accumulation
of osmolytes (GB and proline) and AsA increased the K?/
Na? ratio, and more accumulation of beneficial nutrients in
the roots and leaves enhanced water uptake from soil and
reduced the negative effects of Na? in the salt-stressed
maize plants. The results suggested that foliar-applied AsA
either accelerated the tissue-specific translocation of AsA
& Qasim Ali
qasimbot_uaf@yahoo.com
1
Department of Botany, Government College University,
Faisalabad 38000, Pakistan
2
Department of Physics, University of Agriculture,
Faisalabad 38040, Pakistan
•
or altered the de novo synthesis of AsA to mitigate the
adverse effects of salinity in maize plants.
Keywords Ascorbic acid  Foliar spray  Ion
homeostasis  Osmolytes  Salt tolerance  Water relations
Introduction
Soil salinity severely affects crop productivity, particularly
in the arid and semi-arid regions of the world. The fore-
most effect of salinity is the osmotic imbalance due to the
accumulation of ions, particularly Na? and Cl- in the
rooting medium that severely affects the water uptake
ability of roots and subsequently the translocation to the
dividing cells (Boudsocq and Laurière 2005). Impaired
water uptake also alters the accumulation and translocation
of essential nutrients required for growth, development,
and reproduction of plants (Malash et al. 2011; Ghrab et al.
2013). Higher accumulation of Na? and Cl- causes
specific ion toxicity, and thus alters metabolism, growth,
and production in the plants (Tavakkoli et al. 2011; Suarez
and Grieve 2013).
The phenomenon of osmotic adjustment is considered as
most important for salinity tolerance, during which a net
decrease in cell osmotic potential takes place due to the
accumulation of intracellular solutes without the loss of
cellar water (Blum et al. 1996). Normally, it takes place
through the accumulation of inorganic or organic solutes
such as proline, GB, free amino acids, sugars, polyphenols,
and polyamines (Hoque et al. 2007). In this regard, organic
solutes not only actively take part in the osmotic adjust-
ment but also play an important role for the protection of
cellular enzymes in the presence of high electrolytes. Of all
osmolytes, proline and GB are considered to play an
123

important role in the osmotic adjustments of plants when
under stress (Iqbal et al. 2008). Furthermore, numerous
studies depicted the role of GB in the protection of pho-
tosynthetic machinery, its enzymes (RuBisco) from photo-
damage, and stabilization of cellular membranes under
stressed conditions (Sakamoto and Murata 2002).
Similarly, proline in addition to its role in the osmotic
adjustments regulates the cellular redox potential, acts as
scavenger of hydroxyl radical as well as storage compound
for nitrogen (Sairam et al. 2002). However, different cul-
tivars of the same species even differ in terms of osmotic
adjustment capacity and with respect to the nature of major
solutes taking part in the cellular osmotic potential (Rhodes
and Samaras 1994). Furthermore, the phenomenon of os-
motic adjustment also varies by the degree of stress, plant
age, and by the type of plant part (Shangguan et al. 1999).
For example, de Lacerda et al. (2003) found more accu-
mulation of proline in the shoots as compared with roots in
the salt-stressed plants of sorghum. Similarly, higher ac-
cumulation of organic solutes (proline and GB) was found
in the apical mature leaves of salt-stressed cashew plants
(Viégas and da Silveira 1999). In maize, higher accumu-
lation of proline was recorded in the growing regions of
root and shoot under stressed conditions (Verslues and
Sharp 1999). However, there is lack of literature about the
AsA-mediated plant organ-specific responses in relation to
the endogenous accumulation of AsA and other metabo-
lites in the plants subjected to salinity stress.
In parallel with genetic engineering and conventional
breeding, the induction of stress tolerance has also been
made through exogenous use of various organic and inor-
ganic chemicals (Ali and Ashraf 2011a, b; Iqbal and Ashraf
2013a, b). Ascorbic acid (AsA), commonly known as vi-
tamin C, is involved in the regulation of many important
biological processes such as cell cycle, cell elongation,
enzymatic reactions, biosynthesis of growth regulators, and
photo-inhibition (Dolatabadian and Jouneghani 2009;
Gallie 2012, 2013; Ivanov 2014; Venkatesh and Park
2014). Dolatabadian and Jouneghani (2009) reported that
exogenous application of 100 mM AsA reduced the ad-
verse effects of salt stress in common bean. Foliar appli-
cation of AsA improved the growth, proline accumulation,
and antioxidative enzyme activities in the Saccharum spp.
under salt stress (Ejaz et al. 2012). Similarly, Athar et al.
(2008) reported alleviatory effects of foliar spray of AsA in
wheat under NaCl stress. In cowpea, the induction of salt
tolerance by foliar-applied ascobin (a source of AsA and
citric acid) was associated with the increased accumulation
of GB and proline (Abdelgawad 2014). However, there is a
lack of knowledge about the tissue-specific effects of ex-
ogenous AsA, particularly on the ionic uptake and their
accumulation patterns in relation to the endogenous tissue
123
S. Jamil et al.
AsA concentrations. In the present study, it was hy-
pothesized that foliar-applied AsA could modulate the
uptake of nutrients, accumulation of potential osmolytes
such as proline and GB in different plant parts and plant
water relations in the salt-stressed maize plants. Therefore,
the aim of the present study was to assess whether foliar-
applied AsA could alter plant water relations by altering
the biosynthesis of GB and proline, ionic uptake, and their
accumulation in different plant parts in relation with tissue-
specific AsA accumulation in the salt-stressed maize
plants.
Materials and methods
The seeds of two high yielding maize cultivars (Agaiti-
2002 and EV-98) were obtained from Maize and Millet
Research Institute Yousafwala, Sahiwal, Pakistan. These
cultivars have been widely used in Pakistan for maize
breeding programs due to their better yield and high nu-
tritional quality (Ali et al. 2012). The surface-sterilized
seeds (10) were sown in pots filled with sand treated with
half strength Hoagland’s nutrient solution. After 5 days of
germination, salinity was gradually increased to 120 mM
in one group, while the other group (non-treated plants)
served as control. In the 1st step, 50 mM NaCl solution
prepared in Hoagland’s nutrient solution was applied. In
the 2nd step, after 3 days of first salt treatment, 100 mM
NaCl in Hoagland’s nutrient solution was applied. Finally,
after 5 days of 2nd salt treatment, 120 mM NaCl solution
in Hoagland’s nutrient solution was applied. In all the
cases, excessive solution was added to leach down the al-
ready available salts. In parallel to the saline group, the
control plants were supplied with only Hoagland’s nutrient
solution at each step. After 10 days of final salt treatment,
the plants were sprayed with two levels of AsA (0,
100 mM) supplemented with surfactant Tween-20 (0.1 %).
After 15 days of AsA foliar spray, the data for various
attributes were collected. After estimating root and shoot
fresh weights, the plants were kept in an oven at 70 C for
48 h and the root and shoot dry weights were determined.
Plant water relations
Leaf relative water content
Fully developed young top leaves were used to determine
leaf relative water contents (LRWC). Following equation
was used to calculate the LRWC:
Leaf fresh weight  Leaf dry weight
LRWC ¼  100:
Leaf turgid weight  Leaf dry weight
Modulations in plant water relations and tissue-specific osmoregulation…
Plant water potential, osmotic potential, and turgor
potential
Leaf water potential (Ww) was measured from 7 a.m. to 8
a.m. using a Scholander-type pressure chamber. The leaf
osmotic potential (Ws) was determined using a Vapor
Pressure Osmometer (Wescor 5500, USA) following Ali
and Ashraf (2011a). The difference between leaf Ws and
Ww values was considered as leaf turgor potential.
Biochemicals
Determination of AsA concentration
The AsA concentration in different plant parts was deter-
mined following the method of Mukherjee and Choudhuri
(1983). Five hundred milligram of fresh leaf material was
homogenized in 10 ml of 6 % TCA (w/v). After filtration,
4 ml of the extract was reacted with 2 ml of 2 % acidic
dinitrophenylhydrazine. One drop of alcoholic thiourea
was added to the mixture and boiled in a water bath for
20 min. Then 5 ml of 80 % H2SO4 was added to the ice-
cold mixture. The absorbance of the supernatant was read
at 530 nm using a spectrophotometer (U-4000, Hitachi,
Japan). The concentration of AsA was calculated from a
standard curve using a series of standard solutions
(50–300 mg/l) of AsA (Sigma Aldrich, Germany).
Proline and glycinebetaine determination
The protocol of Bates et al. (1973) was used for appraising
the leaf proline content from fresh leaves. The glycinebe-
taine (GB) from dry leaves was estimated following the
method of Grieve and Grattan (1983).
Determination of minerals
The plant sample (100 mg) was digested using the diges-
tion mixture and the final volume (50 ml) was maintained
in a volumetric flask. Different cations like Na?, Ca2?, and
K? contents were determined using an atomic absorption
spectrophotometer (Hitachi, Model 7JO-8024, Tokyo, Ja-
pan). The nitrogen (N) contents were estimated by a micro-
Kjeldahl method (Bremner and Keeney 1965). The phos-
phorus (P) content was estimated spectrophotometrically
by using Barton’s reagent.
Statistical analysis
The analysis of variance (ANOVA) of the data was done
by using a computer software MSTAT-C (MSTAT
Development Team 1989). The Duncan’s New Multiple
Range Test at 5 % level of probability was used to test the
differences among mean values (Steel and Torrie 1986).
The correlation coefficient analysis among different at-
tributes was performed by Microsoft Excel program and
the principle component analysis (PCA) test was done
using XLSTAT statistical software.
Results
A significant (P B 0.001) reduction in the shoot and root
length was recorded due to the imposition of salt stress in
the rooting medium in both maize cultivars. Salt-induced
reduction in the shoot length was relatively more in the cv.
EV-1098 as compared with cv. Agaiti-2002, but the op-
posite was true for root length. Exogenous application of
AsA significantly reduced the adverse effects of salt stress
on the shoot length of both maize cultivars, however, such
ameliorating effects of foliar-applied AsA were not found
significant on the root length of both maize cultivars
(Fig. 1).
A significant (P B 0.001) reduction in the number of
leaves and plant leaf area was recorded due to root zone
salinity in both maize cultivars. Salt-induced reduction in the
two attributes was more in the cv. EV-1098 as compared
with Agaiti-2002. However, foliar-applied AsA was found
effective in reducing the adverse effects of salt stress on
these growth attributes in both maize cultivars. More in-
crease in the plant leaf area due to the foliar-applied AsA was
evident in the cv. Agaiti-2002 when under salt stress (Fig. 1).
Salt stress significantly (P B 0.001) decreased shoot
fresh and dry weights of both maize cultivars. Foliar ap-
plication of AsA was found effective in reducing the ad-
verse effects of salt stress on shoot fresh and dry weights of
maize plants. More amelioration in shoot fresh and dry
weights due to foliar-applied AsA was recorded in the cv.
Agaiti-2002 as compared with cv. EV-1098 (Fig. 1).
Root zone salinity significantly (P B 0.001) reduced the
root fresh and dry weights of both maize cultivars. Foliar-
applied AsA was found effective in reducing the adverse
effects of salt stress on root fresh and dry weights of both
maize cultivars. The response of both maize cultivars in
relation to increase in root fresh weight due to foliar-ap-
plied AsA was similar under salt stress. While the increase
in root dry weights by foliar application of AsA under
saline conditions was more in the cv. Agaiti-2002 as
compared with EV-1098 (Fig. 1).
A significant reduction in leaf relative water content
(LRWC) was recorded due to imposition of salt stress in
the rooting medium in both maize cultivars. More decrease
in LRWC was recorded in the cv. EV-1098 as compared
with cv. Agaiti-2002. Foliar-applied AsA was found ef-
fective in reducing the adverse effects of salt stress on
LRWC of both maize cultivars, however; the response of
123
 S. Jamil et al.

both cultivars to foliar-applied AsA was similar under salt Foliar-applied AsA was found effective in reducing the
stress (Fig. 1). adverse effects of salt stress on these water relations at-
A significant reduction in plant water potential (Ww), tributes in both maize cultivars, particularly in the cv.
osmotic potential (Ws) and turgor (Wp) of both maize cul- Agaiti-2002 (Fig. 2).
tivars was recorded due to imposition of salt stress. Both A significant increase in GB content due to salt stress
maize cultivars showed similar decreasing trend in these was recorded in all studied plant parts of both maize cul-
water relation attributes due to imposition of salt stress. tivars. Salt-induced increase in GB contents was more in

Agaiti-2002 EV-1098

30 7 14 LSD 5%=1.40
LSD 5%= 1.54 LSD 5%=0.42

25 6 12

Shoot fresh weight (g/plant)

Number of leaves per plant 5 10
Shoot length (cm)

20
4 8
15
3 6
10
2 4

5 1 2

0 0 0

10 LSD 5%=10.11 1.8 LSD 5%=0.08

LSD 5%=0.66 80
9 1.6
70
Plant leaf area (cm 2 )

8
Shoot dry weight (g/plant)
1.4
60
7
Root length (cm)

1.2
6 50
1
5 40
0.8
4
30
0.6
3
20 0.4
2
1 10 0.2
0 0 0

3 LSD 5%=0.23 0.6 LSD 5%=0.05 92 LSD 5%=2.81

90
Leaf relative water content (%)

2.5 0.5
Root fresh weight (g/plant)

Root dry weight (g/plant)

88
2 0.4
86
1.5 0.3
84
1 0.2
82

0.5 0.1 80

0 0 78
0 100 0 100 0 100 0 100 0 100 0 100
mM0 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM
Control Salinity Control Salinity Control Salinity

Fig. 1 Different growth attributes and leaf relative water content of two maize (Zea mays L.) cultivars as influenced by exogenously applied
ascorbic acid (AsA) under control (0 mM) and saline (120 mM) conditions at early growth stage (mean ± SE). 0, 100 mM = levels of AsA

123
Modulations in plant water relations and tissue-specific osmoregulation…

Agaiti-2002 EV-1098 Agaiti-2002 EV-1098 Agaiti-2002 EV-1098

35 LSD 5%=5.23 16 12
LSD 5%=0.57 LSD 5%=3.97

Stem GB content ( μmol g -1 F. Wt.)

Root GB content ( μmol g -1 F. Wt.)

Leaf GB content ( μg g -1 F. Wt.) 14
30
10
12
25
8
10
20
8 6
15
6
4
10
4
5 2
2

0 0 0

35 25 LSD 5%=2.50 25 LSD 5%=3.33

LSD 5%=11.01

Root proline content ( μmol g -1 F. Wt.)

Stem proline content ( μg g -1 F. Wt.)
Leaf proline content ( μg g -1 F. Wt.)

30
20 20
25

15 15
20

15
10 10

10
5 5
5

0 0 0

1.4 LSD 5%=0.19 1.4 LSD 5%=0.23 0.14 LSD 5%=0.038

1.2 1.2 0.12

1 1 0.1
Ψw (-M.Pa)

Ψs (-M.Pa)

Ψp (-M.Pa)

0.8 0.8 0.08

0.6 0.6 0.06

0.4 0.4 0.04

0.2 0.2 0.02

0 0 0
0 100 0 100 0 100 0 100 0 100 0 100
mM mM mM mM mM mM mM mM mM mM mM mM
Control Salinity Control Salinity C ontrol Salinity

Fig. 2 GB, proline, and water relations of two maize (Zea mays L.) cultivars as influenced by exogenously applied ascorbic acid (AsA) under
control (0 mM NaCl) and saline (120 mM NaCl) conditions at early growth stage (mean ± SE). 0, 100 mM = levels of AsA

the cv. Agaiti-2002 as compared with cv. EV-1098. Foliar- Leaf proline content of both maize cultivars was also
applied AsA significantly affected the GB accumulation in increased due to imposition of salt stress but the increase
different plant parts but this accumulation was cultivar and was non-significant. However, the exogenous application
plant part specific. In the leaf, a significant while in the of AsA significantly increased the leaf proline content in
stem a non-significant increase in GB content due to AsA both maize cultivars under salt stressed conditions. Both
application was recorded in both maize cultivars under maize cultivars showed a similar increasing trend in leaf
salinity. However, in the root, AsA application increased proline due to AsA foliar application (Fig. 2).
GB contents under salt stress but only in the cv. EV-1098 Like leaf proline content, stem and root proline contents
(Fig. 2). in both maize cultivars were also increased significantly

123
 S. Jamil et al.

Agaiti-2002 EV-1098 cv. Agaiti-2002 as compared with cv. EV-1098. Foliar-

Leaf AsA content (mg/kg f.wt.)
Stem AsA content (mg/kg f. wt.)
300 LSD 5%=13.76 applied AsA did not affect stem proline contents in both
maize cultivars. However, in the root, foliar application of
250 AsA significantly reduced the proline content in the cv.
Agiti-2002 (Fig. 2).
200 Leaf and stem AsA contents of both maize cultivars
decreased significantly (P B 0.001) due to rooting medium
150 salinity. More decrease in leaf and stem AsA contents due
to salinity were recorded in the cv. EV-1098 as compared
100 with Agaiti-2002. Foliar-applied AsA was found affective
in increasing the AsA contents in the both plant parts of
50 maize cultivars. More increase in leaf and stem AsA con-
tents due to its foliar application under saline conditions
0 were recorded in the cv. EV-1098 as compared with the
cultivar Agaiti-2002 (Fig. 3).
350 LSD 5%=16.66 Root zone salinity significantly (P B 0.001) increased
root AsA content in both maize cultivars. The increase in
300

root AsA content due to salinity was more in the cv. Agaiti-
250 2002 as compared with the cv. EV-1098. Foliar-applied AsA
further increased the root AsA content in both maize culti-
200 vars under saline conditions. More increase in the root AsA
contents due to its foliar application was recorded in the cv.
150
EV-1098 as compared with the cv. Agaiti-2002 (Fig. 3).
100
Imposition of salt stress significantly reduced the K?
contents in leaf, stem, and root of both maize cultivars.
50 Foliar-applied AsA found effective in reducing the adverse
effects of salt stress on K? contents of all plant parts except
0 stem that remained un-effected in this respect. Both maize
cultivars showed a similar increasing trend in case of K?
350 LSD 5%=17.82 contents in the leaf and root due to foliar-applied AsA
(Fig. 4).
Root AsA content (mg/kg F. Wt.)

300
Rooting medium salinity significantly increased Na?
250 contents in the leaf, stem, and root of both maize cultivars,
particularly in the cv. Agaiti-2002, but the opposite was
200
true for stem Na? content. Foliar-applied AsA found ef-
150 fective in reducing the accumulation of Na? in all plant
parts except stem in both cultivars. Furthermore, this AsA-
100 applied decrease in leaf and root Na? was similar in both
maize cultivars (Fig. 4).
50
Imposition of salt stress significantly reduced the K?/
0 Na? ratio in the leaf, stem, and root of both maize culti-
0 100 0 100
mM0 100
mM 0
mM 100
mM vars. However, both maize cultivars showed a similar de-
Control Salinity
creasing trend in case of K?/Na? ratio in all the studied
plant parts. Foliar-applied AsA slightly increased the K?/
Fig. 3 Ascorbic acid contents in different parts of two maize (Zea Na? ratio in the leaf and root of both maize cultivars but
mays L.) cultivars as influenced by exogenously applied ascorbic acid the stem K?/Na? ratio remained un-effected (Fig. 4).
(AsA) under control (0 mM NaCl) and saline (120 mM NaCl) A significant (P B 0.001) reduction in the Ca2? contents
conditions at early growth stage (mean ± SE). 0, 100 mM = levels
of AsA of all plant parts was observed in both maize cultivars
when under salt stress. Maize cultivars did not differ in
relation to this attribute under salt stress. Foliar-applied
due to imposition of salt stress. In the stem, salt-induced AsA significantly increased Ca2? contents in the leaf and
increase in the proline was similar in both maize cultivars root of both maize cultivars and this AsA-applied increase
but in the root, more increase in proline was recorded in the in Ca2? content in these plant parts was similar in both

123
Modulations in plant water relations and tissue-specific osmoregulation…

Agaiti-2002 EV-1098

50 LSD 5%=2.70 50 LSD 5%=2.96 45 LSD 5%=2.85

45 45 40
40 40 35
Leaf K + (mg/g d. wt.)

(mg/g d.wt.)

Root K + (mg/g d.wt.)

35 35 30
30 30
25
25 25
20
20 20

+
Stem K
15
15 15
10 10 10

5 5 5
0 0 0

30 30 30 LSD 5%=0.90
LSD 5%=1.32 LSD 5%=1.67

25 25 25
(mg/g d.wt.)
Leaf Na + (mg/g d.wt.)

Root Na + (mg/g d.wt.)

20 20 20

15 15 15
+
Stem Na

10 10 10

5 5 5

0 0 0

18 LSD 5%=0.34 LSD 5%=0.45 LSD 5%=0.28

30 16
16
14
14 25
12
leaf K +/Na + ratio

Root K +/Na + ratio

Stem K +/Na + ratio

12
20 10
10
15 8
8
6 6
10
4 4
5
2 2
0 0 0
0 100 0 100 0 100 0 100 0 100 0 100
mM mM mM mM mM mM mM mM mM mM mM mM
Control Salinity C ontrol Salinity C ontrol Salinity

Fig. 4 K?, Na? and K?/Na? ratios in different parts of two maize (Zea mays L.) cultivars as influenced by exogenously applied ascorbic acid
(AsA) under control (0 mM NaCl) and saline (120 mM NaCl) conditions at early growth stage (mean ± SE). 0, 100 mM = levels of AsA

maize cultivars. However, foliar-applied AsA did not affect stem and root to foliar-applied AsA. However, the leaf N
the stem Ca2? content (Fig. 5). remained un-affected due to foliar-applied AsA in both
Salinity significantly (P B 0.001) reduced nitrogen cultivars (Fig. 5).
contents of leaf, root, and stem in both maize cultivars. Like N, the P contents in the leaf, root, and stem de-
Both maize cultivars showed a similar decreasing trend in creased significantly (P B 0.001) due to imposition of
case of N contents in all the studied parts. Foliar-applied salinity in the rooting medium. Both maize cultivars
AsA reduced the adverse effects of salt stress on N contents showed a similar decreasing trend in case of P contents in
in the stem and root of both maize cultivars. Both maize all the plant parts in response to salt stress. A non-sig-
cultivars showed similar increasing trend in N contents in nificant effect of foliar-applied AsA was found on the P

123
 S. Jamil et al.

Agaiti-2002 EV-1098
16 14 16
LSD 5%=1.02 LSD 5%=1.15 LSD 5%=1.03
content (mg/g d. wt.) 14 14

content (mg/g d. wt.)

content (mg/g d.wt.)

12
12 12
10
10 10
8
8 8
6
6 6
2+

2+
2+
4
Leaf Ca

Root Ca
4 4

Stem Ca
2 2 2

0 0 0

60 LSD 5%=3.03 50 LSD 5%=3.95 50 LSD 5%=3.71

45 45
50
40 40
Leaf N (mg/g d. wt.)

Root N (mg/g d. wt.)

35 35
40
Stem N (mg/g d. wt.)

30 30
30 25 25
20 20
20
15 15
10 10
10
5 5
0 0 0

8 LSD 5%=0.34 LSD 5%=0.48 7 LSD 5%=0.69

8
7 7 6
Leaf P (mg/g d. wt.)

6
Stem P (mg/g d. wt.)

6 5
Root P (mg/g d.wt.)

5 5
4
4 4
3
3 3
2
2 2
1 1 1

0 0 0
0 100 0 100 0 100 0 100 0 100 0 100
0
mM 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM 0
mM 100
mM
C ontrol Salinity Control Salinity Control Salinity

Fig. 5 Ca2?, N, and P in different parts of two maize (Zea mays L.) cultivars as influenced by exogenously applied ascorbic acid (AsA) under
control (0 mM NaCl) and saline (120 mM NaCl) conditions at early growth stage (mean ± SE). 0, 100 mM = levels of AsA

contents in all the studied plant parts of both maize culti- osmotically active and other organic compounds that trig-
vars (Fig. 5). ger the cellular osmotic adjustments (Barakat 2003). In this
regard, the role of AsA cannot be diminished (Abou-Leila
et al. 2012; Gallie 2013; Ivanov 2014; Venkatesh and Park
Discussion 2014). Normally, the functions of roots and leaves are di-
rectly influenced by the changes in the surrounding envi-
Plants have developed different mechanisms for the better ronment. The plants that are genetically deficit in such
uptake of water under stressful conditions (Munns 2002). compounds could be treated exogenously to counteract
Generally, it takes place through the production of their internal deficiency (Chen and Gallie 2004; Ali and

123
Modulations in plant water relations and tissue-specific osmoregulation…

Table 1 Correlation coefficient values (r2) of AsA contents in dif- Ashraf 2011b). Exogenous application of these compounds
ferent plant parts with growth, different water relation attributes, GB, not only translocated to different plant parts but also trigger
proline, and nutrient uptake in different plant parts
its own biosynthesis in different plant parts (Abdelgawad
AsA L AsA R AsA S 2014; Hameed et al. 2015). However, different plants re-
Leaf AsA 1.000
spond differently to exogenously applied organic com-
Root AsA 0.015ns 1.000
pounds including AsA, possibly due to their differential
Stem AsA 0.927*** -0.038ns 1.000 translocation patterns (Ali and Ashraf 2011b; Hameed et al.
Leaf Ca2? 0.653*** -0.504* 0.776*** 2015). Normally, the functions of roots and leaves are di-
Root Ca2? 0.657*** -0.349ns 0.800*** rectly but differently influenced by the changes in the
Stem Ca2? 0.661*** -0.480* 0.786*** surrounding environment. In the present study, foliar-ap-
Leaf K? 0.634** -0.572** 0.731*** plied AsA was very effective in enhancing its internal
Root K? 0.630** -0.392ns 0.779*** concentrations in different plant parts. The increase in AsA
Stem K? 0.518* -0.710*** 0.640** contents due to its foliar application was more pronounced
Leaf K?/Na? ratio 0.533** -0.706*** 0.657*** in the leaves and roots as compared with the stem. The
Leaf area 0.640** -0.460* 0.639** increase in AsA contents in the leaves and roots shows its
LRWC 0.627** -0.428* 0.722*** phloem-specific translocation as presented earlier by
NL 0.683*** -0.476* 0.799*** Franceschi and Tarlyn (2002). It is known that the stress
NR 0.674*** -0.462* 0.770***
tolerance in plants is the function of combination of all
NS 0.781*** -0.268ns 0.843***
plant parts (leaf, stem and root) (Ali and Ashraf 2011a, b).
Na? L -0.557** 0.702*** -0.652**
However, translocation and accumulation dependent such
Na? R -0.590** 0.647*** -0.689***
effects in stress tolerance of different plants through ex-
Na? S -0.472* 0.737*** -0.575**
NL 0.848*** -0.074ns 0.884***
ogenous use of AsA are still not clear. In the present study,
PL 0.616** -0.515* 0.764***
foliar-applied AsA reduced the adverse effects of salt stress
PR 0.504* -0.399ns 0.697*** and caused a significant increase in plant cellular water
PS 0.687*** -0.499* 0.822*** content that resulted in enhanced biomass production. This
R K?/Na? 0.436* -0.639** 0.616** might be due to the involvement of AsA in the cellular
RDW 0.454* -0.127ns 0.558** osmotic adjustments by the induction of different os-
RFW 0.601** -0.548** 0.718*** molytes accumulation and alternate improvement in the
RL 0.475* -0.560** 0.643** cellular water contents (Athar et al. 2008). Exogenous AsA
S K?/Na? 0.505* -0.695*** 0.649*** has been shown to improve plant growth and yield in dif-
SDW 0.628** -0.568** 0.720*** ferent plants (Athar et al. 2008; Azzedine et al. 2011;
SFW 0.586** -0.402ns 0.688*** Darvishan et al. 2013). In the present study, foliar-applied
SL 0.692*** -0.446* 0.755*** AsA enhanced the accumulation of proline and GB in the
GB L 1.000*** 0.927*** 0.015ns roots and leaves as compared with stem in both cultivars
GB R 0.427* 0.734*** 0.588**
resulting in improvement in the plant water relations at-
GB S -0.592** 0.738*** -0.676***
tributes. More accumulation of GB and proline was asso-
Pro L 0.449ns 0.761*** 0.377ns
ciated with the enhanced endogenous AsA contents in
Pro R 0.636** 0.691*** -0.655***
different plant parts. The parallel increase in the AsA along
Pro S -0.469* 0.825*** -0.561**
WP -0.712*** 0.451* -0.748***
with the GB and proline contents might be attributed to the
SP -0.664*** 0.528* -0.724***
multiple roles of AsA in different biological processes in-
TP 0.681*** 0.446* 0.501* cluding the regulation of enzymatic reactions and synthesis
of different growth regulators (Dolatabadian and Joune-
ns non-significant, AsA ascorbic acid, L K?/Na? leaf K?/Na? ratio,
LA leaf area, LRWC leaf relative water content, N L leaf nitrogen , N R
ghani 2009; Gallie 2012, 2013; Ivanov 2014; Venkatesh
root nitrogen, N S shoot nitrogen, Na L leaf Na?, Na? R root Na?, and Park 2014). The results suggested that AsA-mediated
Na? S stem Na?, NL number of leaves, P L leaf P, P R root P, P S increase in the GB and proline biosynthesis/accumulation
stem P, R K?/Na? root K?/Na?, RDW root dry weight, RFW root might have played an active role in lowering the cellular
fresh weight RL root length, S K?/Na? shoot K?/Na?, SDW shoot
dry weight SFW shoot fresh weight SL shoot length, GB L leaf GB,
osmotic potential that facilitated the water uptake and ul-
GB R root GB, GB S stem GB, Pro L leaf proline, Pro R root proline, timately enhanced the plant growth, representing a possible
Pro S shoot proline, WP water potential, SP solute potentia, TP turgor role of exogenously applied AsA in the cellular osmotic
potential adjustments.
***, ** and * significant at 0.001, 0.01 and 0.05 levels, respectively

123
 S. Jamil et al.

Variables (axes F1 and F2: 92.87 %)

1
Pro-L
PP
AsA-R
0.75
GB-L
AsA-L

AsA-S
0.5
Pro-S

GB-R N-S
Na-S
0.25
Ca-R
Na-L GB-S
Na-R K-R
SFW LRWC
F2 (13.56 %)

Pro-R N-L N-R

Ca-L
0 Ca-S
SP SDW K-L

WP

K-S
-0.25 S-K/Na

-0.5

-0.75

-1
-1 -0.75 -0.5 -0.25 0 0.25 0.5 0.75 1
F1 (79.31 %)

Fig. 6 Principle component analysis showing different associations among different attributes

Imbalance in the availability and uptake of essential
plant nutrients under salinity is a common phenomenon. In
the present study, both cultivars showed differential re-
sponse toward Na? uptake and accumulation. For instance,
more Na? accumulated in the leaves and roots as compared
with stem in both cultivars. Some earlier studies have
shown the significant effects of different organic molecules
on the uptake of Na? in different plants. For example, in
the wheat, Raza et al. (2007) reported that exogenous ap-
plication of GB as foliar spray reduced the uptake of Na?
and increased the K?/Na? ratio. In the present study, foliar
application of AsA was found very effective in reducing
the uptake of Na?, while increasing that of K? and other
essential nutrients in parallel with enhanced accumulation
of GB and proline in both leaves and roots except plant
stem. This low uptake of Na? due to foliar AsA and its
high content in the leaf and root resulted in high K?/Na?
ratios and ultimately caused better biomass production.
Such improvement in plant salt tolerance due to exogenous
AsA has been reported in some earlier studies (Athar et al.
2008, 2009; Dolatabadian and Jouneghani 2009; Farouk
2011; Ejaz et al. 2012). Furthermore, this increase in maize
plant biomass was negatively associated with Na? accu-
mulation in different plant parts especially in the roots and
leaves when under salt stress. The reduction in Na? ac-
cumulation due to AsA application was positively related
with more AsA concentration in the root and leaf as
compared with stem. The higher AsA concentrations in the
leaves and roots could be due to more translocation of AsA
to these plant organs or due to the de novo synthesis of
AsA (Abdelgawad 2014; Hameed et al. 2015). The reduced
Na? accumulation in the leaf and root resulted in increased
K?/Na? in the leaves and roots as compared with stem.
Taken together, the results suggested a significant role of
AsA in the cellular ion homeostasis.
Data presented in the Table 1 and Fig. 6 (supplementary
data) for correlations and principle components analysis
(PCA), respectively, showed a significant positive corre-
lation of AsA concentrations in the leaf and shoot with
various growth attributes and nutrients contents (K?, Ca2?,
N, and P) in different plant parts. The reverse was true in
case of Na? concentration in different plant parts.

123
Modulations in plant water relations and tissue-specific osmoregulation…
However, root endogenous AsA concentrations did not
show such relationships. A positive significant correlation
of AsA was found in the leaf and root with proline
(0.761***, 0.449* and 0.691***, 0.636**) and GB (1.00***,
0.927*** and 0.427*, 0.588**) contents, respectively. The
positive relationships in the roots and leaves suggested
AsA role in the proline and GB metabolism, and thus
cellular osmotic adjustments. A significant positive corre-
lation was also found among K?/Na? ratios and AsA
contents in different plant parts such as in leaf (0.533**,
0.657***), stem (0.505*, 0.649***) and root (0.436*,
0.616**), respectively. A positive significant correlation
was found among AsA content in leaf and stem with the
LRWC (0.627**, 0.722***), respectively. The correlations
and PCA analysis suggested that an increase in the AsA
contents in leaf and root due to its foliar application
showed inhibitory effects on Na? uptake that increased
K?/Na? ratios and resulted in the induction of salt toler-
ance in the maize plants along with increased biomass
production.
Conclusions
Based on the results of the present study, it could be con-
cluded that AsA-induced salt tolerance in the maize plants
is the function of its translocation/de novo synthesis in the
specific plant parts whereby altering the plant water status
and cellular osmotic adjustments possibly through the en-
hanced accumulation of proline and GB and enhanced
uptake of beneficial nutrients. Subsequently, these benefi-
cial changes increased growth of maize plants. The AsA
accumulation caused a significant reduction in the uptake
of Na? and thus its accumulation in the leaf and root that
alleviated the deleterious effects of Na? on plant growth.
The results are helpful in advancing our knowledge about
the specific involvements of AsA in the tolerance of plants
grown under salinity regimes.
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