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Chapter 4 Discussion
177
4. Discussion
Availability of water is one of the most important factors, which determine
geographical distribution and productivity of plants (Bartels, 2001). Water stress
is perceived as water deficit and can occur with different severity (Ramanjulu
and Bartels, 2002). A continuation of a mild water deficit leads to drought and
even desiccation (loss of most of the protoplasmic free or bulk water). The
response and adaptation of plants to such conditions are very complex and
highly variable. Being sessile organisms, plants have developed various
strategies to acquire stress tolerance. These strategies include changes in
metabolic processes, structural changes of membranes, expression of specific
genes and production of secondary metabolites (Shinozaki and Yamaguchi-
Shinozaki, 2000; Ramanjulu and Bartels, 2002). In a word, the study of the
physiological mechanisms of wheat anti-drought has much work to do (Shao et
al.; 2008).
4.1. Shoot Growth, Morphological Characteristics, Pigments Content and
Some Metabolites
Growth is influenced by various internal and external factors besides its
genetic makeup and is an important tool for assessing crop productivity in
various crops. The aforementioned pattern of results revealed that, drought
stimulates root growth by increasing root length, number of adventitious roots
and root plasticity. On the other hand, root fresh and dry masses appeared to
decrease in response to water stress. Furthermore, water stress led to a drastic
decrease in shoot growth vigor (shoot length, plant height, fresh and dry masses,
shoot diameter and leaf area). The inhibitory effect of water stress was more
pronounced on the sensitive cultivar than on the resistant ones. The variation in
Chapter 4 Discussion
178
response of wheat cultivar for stress (drought and salt) tolerance was known in
many studies (Iqbal and Ashraf, 2005; Arfan et al., 2007). In this respect Sankar
et al. (2007), have recoded that the root length, shoot length, total leaf area,
fresh weight and dry weight of bhendi were significantly reduced under drought
stress treatment.
Continuation of root growth under drought stress through stimulation in root
length and number of adventitious roots is an adaptive mechanism that
facilitates water uptake from deeper soil layers. These results were in
accordance with those obtained by (Sundaravalli et al., 2005; Yin et al., 2005).
Furthermore, arrest of plant growth during stress conditions largely depends
on the severity of the stress. Mild osmotic stress leads rapidly to growth
inhibition of leaves and stems, whereas roots may continue to elongate (Spollen
et al., 1993). The degree of growth inhibition due to osmotic stress depends on
the time scale of the response, the particular tissue and species in question, and
how the stress treatment was given (rapid or gradual).
Morphological characters like fresh and dry masses have a prefund effect in
water-limited conditions (Shao et al., 2008). In this investigation, the reduction
in fresh and dry masses in root, shoot and flag leaves of droughted wheat
cultivars may be responsible for the suppression of plant growth and
consequently affected crop productivity (Tables 1, 2a & 2b). Overall, there is a
sharp contrast between the root and the shoot in their response to water deficit. It
could be explained by different rate of osmotic adjustment of shoot and root
cells (Hsiao, 2000) or various loosening ability of leaf cell walls from roots cell
walls. Loosening ability of the growing cell wall could be affected by auxins and
also by ABA. Under water stress, the concentration of endogenous ABA
increases in both leaves and roots and more ABA is transported from root to the
shoot (Davies and Zhang, 1991). Simultaneously, convincing evidence was
Chapter 4 Discussion
179
obtained indicating that ABA maintains root growth while inhibiting shoot
growth in soybean (Creelman et al., 1990) and in maize (Saab et al., 1990) at
low water potential conditions.
The root/shoot ratio increases under water stress conditions to facilitate water
absorption (Nicholas, 1998). The growth rate of wheat and maize roots was
found to decrease under moderate and high water-deficit stress (Noctor and
Foyer, 1998). However, the development of the root system increases water
uptake and maintains the right osmotic pressure through higher proline levels
(Pan et al., 2003). An increased growth was reported in mango under water
stress (Jaleel et al., 2007a). The root dry weight decreased under mild and severe
water stress in sugar beet (Pan et al., 2002). A significant decrease in root length
was reported in water stressed Populus species by (Panda and Khan, 2003).
Results in tables 2a and figure15 indicated that, water stress caused noticeable
decreases in the shoot length and plant height. In fact the reduction in plant
height may probably due to the decline in the cell enlargement that result from
low turgor pressure and more leaf senescence under water stress (Manivannan et
al., 2007b). Also, water stress is a very important limiting factor at the initial
phase of plant growth and establishment (Shao et al., 2008). There was a
significant reduction in shoot height in Populus cathayana under deficit stress
(Nautiyal et al., 2002). In soybean, the stem length decreased under water-
deficit stress, but this decrease was not significant when compared to well-
watered control plants (Shao et al., 2008).
Reduction in leaf area by water stress is an important cause of reduced crop
yield through reduction in photosynthesis (Rucker et al., 1995). The growth
retardation in leaf area of droughted wheat plants (Table 2b & Fig. 17) is mainly
due to water stress decreased the turgor (see 4.4) which may diminish both cell
Chapter 4 Discussion
180
production and cell expansion within the leaves. These results were in a good
agreement with those obtained by Hsiao (2000); Choluj et al. (2004).
Rolling of leaves was observed to occur mainly in the susceptible wheat
cultivar. There are two possible ways in which a plant in a droughted
environment may benefit from rolling its leaves. Firstly, damage by increased
leaf temperature resulting from high levels of solar radiation incident on leaf
surfaces could be minimized by reducing the effective leaf area presented to the
sun's rays, so that less radiation is intercepted by leaf tissue (Begg, 1980).
Secondly, transpiration rates could be reduced through the creation, by leaf
rolling, of a microclimate with both higher humidity and boundary layer
resistances near the leaf surfaces, thereby conserving scarce water resources
(Oppenheimer, 1960).
The applied chemical appeared to improve the growth vigor by increasing the
plant height, fresh and dry masses, and leaf area and root length of both wheat
cultivars. The protective role of GB on wheat growth can be related to its role in
osmotic adjustment where it acts as a non-toxic cytoplasmic osmolyte (Ibrahim
and Aldesuquy, 2003). In addition, the beneficial effect of GB application on
droughted wheat plants may probably be due to GB induced production
additional vacuoles in root cells, which resulted in a greater accumulation of Na+
in the root and a decrease in its transportation to the shoot (Lutts et al., 1999). In
this respect, Demiral and Türkan (2004) studied the ability of exogenously
applied glycine betaine for the alleviation of growth inhibition and senescence
resulting from NaCl stress. They found that shoot fresh weight of Pokkali and
shoot and root dry weight of IR-28 showed a decrease under salinity but an
increase with exogenous GB application. In addition to its direct protective
roles, either through positive effects on enzyme and membrane integrity or as an
osmoprotectant, GB may also protect cells from environmental stresses
Chapter 4 Discussion
181
indirectly via its role in signal transduction. For example, GB may have a role in
Na+/K
+ discrimination, which substantially or partially contributes to plant salt
tolerance. Ion homeostasis in plants is governed by various membrane transport
systems (Ashraf and Foolad, 2007).
Exogenous application of SA had a promotive effect on growth vigor of root,
shoot and leaf area of both wheat cultivars under non-stress and stress
conditions. These results can be related to earlier studies which observed that
exogenous application of SA promotes growth and counteracts the stress
induced growth inhibition due to abiotic stresses in a range of crop species (Tari
et al., 2002; Shakirova et al., 2003; Singh and Usha, 2003; Khodary, 2004; El-
Tayeb, 2005b; Arfan et al., 2007). For example, salinity stress-induced growth
inhibition was alleviated by exogenous SA application through the rooting
medium on the growth of tomato (Tari et al., 2002) and Phaseolus vulgaris
(Stanton, 2004). Similarly, foliar spray with SA also mitigated the adverse
effects of salt stress on growth of maize (Khodary, 2004) or promoted the
growth in soybean (Gutierrez-Coronado et al., 1998). Singh and Usha (2003)
reported that foliar spray with SA counteracted growth inhibition in wheat
caused by water stress, one of the major factors caused by salinity stress in
plants. Salicylic acid-induced increase in growth of wheat under non-saline or
saline conditions can be attributed to an increase in photosynthesizing tissue,
i.e., leaves (Dhaliwal et al., 1997; Zhou et al., 1999), which is in agreement with
our results.
Gutierrez–Coronado et al. (1998) observed significant effect of SA on soybean
increases in shoot growth, root growth and plant height. Also, Khodary (2004)
reported that SA increased the fresh and dry weight of shoot and roots of salt
stressed maize plants. Furthermore, Sawada et al. (2008) recorded that salicylic
acid (SA) accumulates in salt-stressed rice (Oryza sativa L. cv. Nipponbare)
Chapter 4 Discussion
182
seedlings and they hypothesized that the accumulation of SA might potentate
oxidative injury in rice seedlings since the inhibition of SA synthesis alleviated
the growth inhibition under high salinity.
The prevention of water loss from leaves of droughted wheat plants as results
of SA application might be the principal reason for the significant increase in
shoot fresh weight. The observed increase in growth of droughted wheat plants
resulting from SA (antitranspirant) may due to its effect on improvement of
turgidly at a time when the growth of that particular plant part was more
dependent on water status than in photosynthesis (Davenport et al., 1972).
Increasing the efficiency of photosynthesis has long been a goal of plant
research (Na´tr and Lawlor, 2005). The site of the photosynthesis in plants is
directly depends upon the chlorophyll bearing surface area, irradiance and its
potential to utilize CO2 (Hirose et al., 1997). Photosynthesis is a key metabolic
pathway in plants. In fact, maintaining good photosynthetic rate leads to
maintenance of growth under water stress (Dubey, 2005). The present results
indicated that water stress caused marked decreases in the pigment contents in
the flag leaves of the wheat plants (Table 3 and Figures 18&19). There is a
common observation that leaf yellowing can occur when leaves have had low
water potentials for a considerable time. Chlorophyll is more sensitive to
drought than carotenoids and consequently the ratio of total chlorophyll to
carotenoids decreases with increasing drought severity (Barry et al., 1992).
In accordance with these results, Manivannan et al. (2007c) found that the
water deficit affected the early growth, biomass allocation and pigment of five
varieties of sunflower (Helianthus annuus L.) plants. They found that there was
a significant difference in early growth, dry matter accumulation and pigment
among the studied varieties. The root length, shoot length, total leaf area, fresh
and dry weight, chlorophyll a, b, total chlorophyll and carotenoids were
Chapter 4 Discussion
183
significantly reduced under water stress treatments. Moreover, Kamel et al.
(1995) working with cotton plants (Gossypium barbadense cv Giza 75) reported
two opposite trends regarding the relative concentration of photosynthetic
pigments throughout the experimental period in plants under constant water
stress. The first was a reduction in different photosynthetic pigments comparing
to the normally irrigated plants, while the second was an enhancing effect.
Subjecting cotton plants to drought whether during the vegetative growth or at
maximum flowering, showed a decrease in the concentration of chlorophylls a
and b and carotenoids. However, at budding or flowering a remarkable increase
occurred compared with controls.
It is clear that, the decline in pigments (chl a, chl b and carotenoids) content
in flag leaves of sensitive and resistant cultivates under drought, may accelerated
the ageing process in the sensitive cultivar more than in the resistant ones. The
stimulating effect of GB on photosynthetic pigments may probably be due to GB
had a protective role on photosynthetic apparatus of sensitive cultivar and
decrease the rapid senescence of flag leaves occurred as a result of water stress
(Mäetak el al., 2000).
In general, SA application induced noticeable increases in pigments content
(chl a, chl b and carotenoids) in flag leaves of droughted wheat cultivars (Table
4). The stimulating effect of SA may be due to the fact that SA led to increase
leaves longevity on droughted plants by retaining their pigments content,
therefore inhibit their senescence. In relation to these results, Chandra and Bhatt
(1998) observed that an increasing or decreasing effect of SA on chlorophyll
content of cowpea (Vigna unguiculata) depends on the genotype. In another
study, treatment with SA increased pigment contents in soybean (Zhao et al.,
1995), maize (Khodary, 2004), and wheat (Singh and Usha, 2003; Arfan et al.,
2007) grown under normal or stress conditions. Futthmore, Arfan et al. (2007)
Chapter 4 Discussion
184
found that the improvement in growth and grain yield of wheat salt-tolerance
due to SA application was associated with improved photosynthetic capacity.
Carbohydrates that represent one of the main organic constituents of the dry
matter were found to be affected by water stress. In this study, glucose, sucrose
and total soluble sugars were increased in the flag leaves of two wheat cultivars
at heading and anthesis (Table 4 & Figure 20). This is consistent with the widely
observed of increase soluble sugars in response to water stress in both resistant
and sensitive plants (Al-Hakimi et al., 1995; Ibrahim, 1999). Drought stress
decreases photosynthetic rate and disrupts carbohydrate metabolism in leaves
(Kim et al., 2000); both may lead to a reduced amount of assimilate available for
export to the sink organs, and thereby increasing the rate of reproductive
abortion.
Liu et al. (2004) studied the effect of drought stress on carbohydrate
concentration in soybean leaves and pods during early reproductive
development. They found that drought did not affect the activity of soluble
invertase in leaves. In flowers and pods, sucrose concentrations were higher
under drought as compared with well-watered controls. Hexose and starch
concentrations of flowers and pods were also higher under drought; thereafter
they were significantly lower than those of the well-watered controls. Soluble
invertase activity was decreased by drought in pods. The total amount of non-
structural carbohydrate accumulated in pods during the sampling periods was
substantially reduced by drought.
Drought stress also reduces photosynthesis, for a number of reasons: i)
hydroactive stomatal closure reduces the CO2 supply to the leaves, ii) water
deficiency damages the cytoplasm ultrastructure and enzyme activity, iii)
dehydrated cuticles, cell walls and plasma membranes are less permeable to
Chapter 4 Discussion
185
CO2. An analysis of the correlation between drought and the carbohydrate
metabolism reveals that one characteristic symptom of water deficiency is the
mobilization of the starch stored in the chloroplasts. As there is also a reduction
in the translocation of carbohydrates during drought stress, this leads to a change
in source-sink relationships (Liu et al., 2004).
Growth arrest resulted from water withholding can be considered as a possibility
to preserve carbohydrates for sustained metabolism, prolonged energy supply,
and for better recovery after stress relief (Bartels and Sunkar, 2005). The
inhibition of shoot growth during water deficit is thought to contribute to solute
accumulation and thus eventually to osmotic adjustment (Osorio et al., 1998).
For instance, hexose accumulation accounts for a large proportion of the osmotic
potential in the cell elongation zone in cells of the maize root tip (Sharp et al.,
1990).
As sucrose is both the principal and the preferred form of photosynthate for
long-distance transport to sink organs, its concentration in leaves should
represent the current availability of assimilate for reproductive development
(Westgate and Grant, 1989). The results also indicated that the increase in the
sucrose with a concomitant increase with glucose content in flag leaves of both
wheat cultivars and this is in conformity with the work of Ibrahim (1999) who
observed that sorghum leaves accumulated sucrose, glucose and total soluble
sugar in response to water stress. It has been suggested that drought induced
sucrose accumulation in crop reproductive organs may be partially due to a low
activity of acid invertase, which fails to cleave the incoming sucrose into hexose
under drought conditions (Andersen et al., 2002). As a result of this, the ratio of
hexose to sucrose, which has been suggested to play an important role in
regulation ovary and seed development (Weschke et al., 2000), may thus be
reduced under drought stress.
Chapter 4 Discussion
186
Results in table 4 and fig. 21 indicated that, polysaccharides and total
carbohydrates contents were decreased in droughted wheat flag leaves. The
sensitive cultivar suffered more reduction than the resistant ones. This is in
agreement with the observed accumulation of starch in sorghum plants by
Ibrahim (1999) in response to water stress. This increase would be advantageous
in terms of carbohydrate reserve for growth e.g. more root (Chaves et al., 1995)
or leaves osmoregulation (Ackerson, 1981). In pigeon pea (Cajanus cajan), leaf
starch and sucrose concentrations decreased rapidly and becomes close to zero,
while the concentrations of glucose and fructose significantly increased in
response to drought stress (Keller and Ludlow, 1993). Similar results have been
observed in several plant species under drought conditions (Lawlor and Cornic,
2002).
Applications of GB (osmoprotectant) induced additional increases in soluble
sugars and stimulate the biosynthesis of polysaccharides and consequently total
carbohydrates in flag leaves of the two wheat cultivars. This was in agreement
with the finding of Ibrahim and Aldesuquy (2003); Ibrahim (2004) who found
that droughted sorghum plants treated with GB accumulated more soluble sugars
than the droughted plants only.
Under water stress conditions, SA-pretreated plants showed significant
increases in the soluble sugars, polysaccharides and total carbohydrates content
than the SA-untreated plants. Similar results were obtained by Khadary (2004)
and El-Tayeb et al. (2006) with sunflower plants under Cu stress. In this respect,
El-Tayeb (2005b) investigated that; total soluble sugars were accumulated in
salt-stressed barley plants treated with 1 mM salicylic acid (SA). Furthermore he
reported that, exogenous application of SA appeared to induce preadaptive
response to salt stress leading to promoting protective reactions to the
photosynthetic pigments and maintain the membranes integrity in barley plants,
Chapter 4 Discussion
187
which reflected in improving the plant growth.
Our results indicated that, water stress led to massive increases in the soluble
nitrogen (amino-N, ammonia-N, amide-N and total soluble nitrogen) but
decreased total nitrogen and soluble protein (Table 5 and Figures 22&23). In
order to increase its ability to overcome water stress, the resistant cultivar was
able to keep out its nitrogen and protein content under water stress at higher
levels than the sensitive ones. These results were in good conformity with that of
Khalil and Mandurah (1990) who studied the effect of water stress on nitrogen
metabolism of cowpea plants. They observed that water stress decreased shoot
total-N and protein-N but increased the soluble-N content. This change in
nitrogen content may be related to the inhibition of translocation from root to
shoot, inhibition of protein synthesis or the increase of protease activity.
The increase of soluble nitrogen compounds are of importance in plant
osmoregulation in response to water deficit. In this respect, Mohammadkhani
and Heidari (2008) found that, the initial increase in total soluble proteins during
drought stress was due to the expression of new stress proteins, but the decrease
was due to a severe decrease in photosynthesis. Photosynthesis decreased in
drought stress (Havaux et al., 1987) and materials for protein synthesis weren’t
provided; therefore, protein synthesis dramatically reduced or even stopped. In
addition, the increase in total soluble proteins under drought stress was
consistent with the findings of Riccardi et al. (1998) and Ti-da et al. (2006) in
maize, and Bensen et al.(1988) in soybean. These authors reported that drought
stress resulted in an increase of some soluble proteins and a decrease of others.
Foliar application of GB caused marked increases in the soluble nitrogen and
enhancing the total nitrogen as well as soluble protein in the droughted wheat
plants of the two cultivars. GB application may lead to increase free amino acids
Chapter 4 Discussion
188
especially proline in the water stressed wheat plants (see 4.5) and consequently
increased the soluble nitrogen as well as total-N. In this respect, Ibrahim (2004)
who found that, in correcting the N concentration to total shoot dry weight found
that, salinity had a negative effect on N content, and GB improved total-N of
salinity stressed sorghum plants.
Salicylic acid treatment led to an accumulation in the soluble nitrogen, the
total nitrogen as well as soluble portion in the flag leaves of droughted wheat
plants of the two cultivars. These results were in consistant with (El-Tayeb et al.
2006). In addition, SA treatment protected nitrate reductase activity and
maintained the protein and nitrogen contents of the leaves, compared to water-
sufficient plants. The results signify the role of SA in regulating the drought
response of plants and suggest that SA could be used as a potential growth
regulator to improve plant growth, under water stress (El-Tayeb et al. 2006).
4.2. Image Analysis for Measuring the Anatomical Features in Flag Leaf
and Peduncle of the Main Shoot
Most plants have developed morphological and physiological mechanisms
which allow them to adapt and survive (Ludlow, 1989). These mechanisms
mainly comprise a reduction of the leaf size, leaf rolling, dense leaf pubescence,
deeply developing stomata, accumulation of mucilage and other secondary
metabolites in the mesophyll cells, increase of mesophyll compactness
(Bosabalidis and Kofidis, 2002).
Results in table 6 and plates 1, 5 indicated that, water stress induced marked
decreases (P< 0.05) in thickness of the leaf and ground tissue in both wheat
cultivars. This is presumably due to a reduction in mesophyll tissue of the flag
leaf. These results were in accordance with those obtained by Aldesuquy et al.
(1998b) with wheat plants under salinity. According to Cutler et al. (1977)
Chapter 4 Discussion
189
reduction in cell size appears to be a major response of cells to water deficiency.
Furthermore, the reduction in cell size under water stress conditions may be
considered as drought adaptation mechanism (Steudle et al., 1977).
Drought stress application with two olive cultivars (Mastoidis and Koroneiki)
resulted in a decrease of the size of the epidermal and mesophyll cells with a
parallel increase of the cell density. These changes were more characteristic in
cv. ‘Mastoidis’. Stomata became more numerous and smaller, while non-
glandular hairs (scales) greatly increased in number particularly in cv. Koroneiki
(Bosabalidis and Kofidis, 2002).
Treatment with GB, SA or their interaction induced marked increases in
thickness of both leaf and ground tissues. This was clear in table 6 and plate
2,3,4,6,7,8 . The increase in the leaf thickness might be due to the increase in the
spongy parenchyma tissue and/or palisade parenchyma tissue. These tissues
(mesophyll) are characterized by high concentration of chloroplast. As the leaf
thickness could be considered as a good indicator to specific leaf weight which
is a reliable index to photosynthetic efficiency. It could be concluded that, the
resistant cultivar was more efficient in synthesizing metabolites than the
sensitive one. In section 3.5 there was a positive relationship between leaf area
and yield of wheat cultivars where, resistant cultivar produce higher yield. These
results were in accordance with Planchon (1969) who showed that high yielding
ability is strongly associated with longer leaves.
The conductive canals between source and sink in wheat cultivars have been
reported to play a prominent role in the translocation of photosynthates to the
developing grains (Evans et al., 1970). Water stress reduced the peduncle
diameter (Table 7 and Plate ). This reduction may a result of decreased cell
enlargement and cell division (Nieman, 1965), or inhibition of meristematic
Chapter 4 Discussion
190
division (El-Kabbia et al., 1981).
As aforementioned in vascular bundle area of flag leaf, smaller area of
vascular bundle of both wheat cultivars may be associated with higher number
in the peduncle section. So the total area of conductive tissue of the peduncle of
the resistant cultivar may exceed that of sensitive one. As shown by Evans
(1970), the area of the phloem tissue of the peduncle of wheat from all stages of
evolution of the crop, varies over ten fold range from wild diploid Aeglops to
modern hexaploid.
The obtained results revealed that, drought caused reduction in metaxylem
vessel and xylem tissue areas in flag leaf and peduncle of both wheat cultivars.
This may probably be due to the decrease in the number of additive divisions in
the cambium (El-Shami, 1987). Furthermore, this would result in a lower rate in
translocation of water necessary for photosynthesis. These results were in
accordance with those obtained by Aldesuquy et al. (1998b) with wheat plants
under salinity.
The applied chemicals induced additional increases in the areas of
conductive canals (xylem and phloem) in flag leaf and peduncle of both
cultivars. In addition, the application of these chemicals caused positive
correlations between grain yield and vascular bundle area, xylem area and
phloem area in both leaf and peduncle of the two cultivars. This furnishes better
translocation of assimilates from flag leaf (as source) towards the developing
grains (as sink) through the conductive canals, where there was a good source-
sink relationship particularly there was positive correlation between phloem
tissue area in leaf and peduncle of both wheat cultivars and assimilates
(polysaccharides, protein and total nitrogen). In this regard, Evan et al. (1970)
motioned that the phloem area was found to be directly proportional to the
Chapter 4 Discussion
191
calculated maximum rate of assimilate import by the ears for the 22 lines of
wheat examined and expressed as rate per cm2 of the phloem area was similar to
rates calculated for import into other rapidly growing organs.
The highly numbers of hairs on the flag leaf surfaces as a result of water
stress treatment may probably due to that trichomes (hairs) may increase the
leaf boundary layer resistance and this may improve water use efficiency. These
results were in a good agreement with (Quarrie and Jones, 1977). Also number
of hairs on the peduncle surface increased as response to drought in both wheat
cultivars. In this connection, Udovenko et al. (1976) found that salinity
treatments increased the number of hairs on peduncle surface as indicated in the
results of this investigation (Table 8 and Plate 9,13). This may support the idea
that hairs have protective action against the reduction of water loss from
peduncle surface. On the other hand, application of GB, SA or their interaction
caused additional increases in the number of hairs. This was in accordance with
Aldesuquy et al. (1998b) with wheat plants treated with sodium salicylate under
salinity.
Water stress showed an increase in the number of closed stomata and
decreases in the number of opened ones in the flag leaf of both wheat cultivars
particularly the resistant ones. This modification appeared to be an adaptive
mechanism toward drought conditions. In this respect, Fahn and Cutler (1992)
reported that, many xerophytes in order to save internal water develop their
stomata in local leaf epidermal depressions or in crypts. Furthermore, they
reported that, a major process of water economy is the reduction of transpiration
by closure of stomata. This process entails in parallel a reduction of the rate of
photosynthesis, since CO2 is prevented to enter the mesophyll. Decline of
photosynthesis in water stressed plants was found, however, not to be
exclusively due to closure of stomata.
Chapter 4 Discussion
192
As compared to control, application of GB (osmoprotectant), SA
(antitranspirant) or their interaction decrease the number of opened stomata on
both upper and lower epidermis in flag leaf and peduncle. Therefore increase the
water content in leaf necessary for photosynthesis. Salicylic acid application
induced an adaptive response to water stress by keeping turgidity of leaves
(relative water content in section 3.4) through stomatal closure.
Water stress increased the number of motor cells on the flag leaf upper
epidermis of both wheat cultivars. Furthermore, treatment with GB, SA or their
interaction added more increase in the number of motor cells in the fag leaf
(Table 6 and Plate 2,3,4,6,7,8). This increase induced an adaptive response of
wheat plants towards drought, where the role of motor cells is important in leaf
rolling. Like muscle cells which have excitation-contraction coupling, the motor
cells in plants exhibit rapid movement resulting from excitation tugor loss
(Sibaoka, 1980). Regarding the mechanism of movement in Mimosa, Sibaoka
(1991) reported that the motor cells contain a fibrillar structure, contraction of
these fibrils may open pores in the membrane of the motor cells upon activation.
Outward bulk flow of the vacuolar sap through these pores, due to the pressure
inside the cell, results in turgor loss of the motor cells and then the bending of
the organ. In fact, the susceptible cultivar contained higher number of motor
cells as a result of GB, SA or their interaction which facilitated the rolling of
leaves in that cultivar than the resistant one. These chemicals appeared to be
benefit in improving tolerance of wheat plants towards water stress.
4.3.Induction Of Stomatal Closure, Reduction of Transpiration,
Improvement Of Leaf Turgidity And Hormonal Regulation
The results of this investigation clearly elucidated that, drought markedly
reduced (P<0.05) the diurnal values of transpiration allover day time of both
Chapter 4 Discussion
193
wheat cultivars (Table 9& Fig. 24). Consequently, there was also marked
depletion in the mean daily values of transpiration (Fig. 27a). The reduction in
transpiration rate appeared mainly to be due to firstly, transpiration rate could be
reduced through the creation by leaf rolling (which minimized the leaf area
subjected to the sun ُ s rays) occurred obviously in the sensitive cultivar than the
resistant one and secondly, the obvious reduction in stomatal area on both upper
and lower surfaces. Moreover, these results were in conformity with those
obtained by El-Sharkawi and Salama (1975) who found reduced soil potential
induced by either moisture deficiency or increased salinity reduced both
transpiration rate and lay turgidity in wheat and barely plants during the day
time and the reduction was furthered by increased water stress. Furthermore,
Bieloria and Mendel (1969) conducted simultaneous measurements of apparent
photosynthesis and transpiration rates and they found that these parameters
decreased with decreased soil moisture.
Glycine betaine, salicylic acid or their interaction caused an additional
decrease in the transpiration rates of both wheat cultivars. According to Ibrahim
and Aldesuquy (2003), water stress remarkably reduced sorghum transpiration
rate and total leaf conductance at both lower and upper leaf sides. Application
with GB, shikimic acid or their interaction caused additional decreases in the
transpiration rates and total leaf conductance.
In the present study, the reduction in stomatal aperture induced by water
stress (Table 10 and Fig. 25, 26) was mainly due to increasing in the
biosynthesis of ABA in wheat flag leaves. This hormone increases the turgor
pressure of the whole plant by inducing stomatal closure. These results were in
accordance with those obtained by Schroeder et al. (2001); Dorothea and
Ramanjulu (2005). In addition, these data are consistent with the finding of
Tardieu and Davies (1992) who observed diurnal variations in the sensitivity of
Chapter 4 Discussion
194
maize stomata to ABA. They provide strong modifies sensitivity, with an
increased stomatal sensitivity to ABA during the afternoon hours when leaf
water potentials are low.
Treatments with GB, SA or their interaction caused an additional decrease in
the transpiration rates and stomatal areas of both wheat cultivars. The effect was
more pronounced with SA and GB+SA treatments. These results were in good
agreement with those obtained by Agboma et al. (1997a) who found that
exogenous application of GB to soybean reduced transpiration rate, under all
conditions, to 85% of untreated plants. In this respect, Aldesuquy et al. (1998a)
studied the effect of antitranspirants (sodium salicylate or ABA) and salinity on
some water relations of wheat plants. They found that sodium salicylate or ABA
appeared to regain the leaf turgidity of saline-treated plants by inducing
additional decreases in transpiration rates through more reduction in stomatal
pores on both upper and lower surfaces of leaf and therefore improved the water
use efficiency.
The pattern of changes in leaf turgidity resulted from water stress condition
appeared also to go in close parallelism with the changes in transpiration rates
and stomatal areas (Table 11 &Fig. 28). This indicates that there is a concrete
relationship between the extent of stomatal opening, transpiration rate, and the
leaf turgidity in wheat plants. The decrease in the leaf turgidity of droughted
wheat plants may be due to insufficient root system to compensate the water lost
by transpiration and/or unavailability of water in the soil. These results were in
agreement with those obtained by (Lawlor and Cornic, 2002).
Application of GB, SA improves the RWC of the wheat plants grown under
water stress conditions. These were the same results obtained by Aldesuquy et
al. (1998a) with wheat plants under the application of salinity and
antitranspirants (sodium salicylate or ABA).
Chapter 4 Discussion
195
A better understanding of the physiological strategy adopted by a drought
resistant variety to cope with water deficit requires through study of the
relationship between WUE and transpiration (Sankar et al., 2008). Thus, the
WUEG and WUEB of the two wheat cultivars were greatly reduced by water
stress and this was clear with the sensitive cultivars more than the resistant one
(Figures 30c, d). This was in agreement with Anyia and Herzog (2004) with
cowpea plants subjected to water stress; Aldesuquy and Ibrahim (2001) with
wheat plants under salinity. However, treatments with GB, SA or their
interaction mitigated the effect of drought on WUEG and WUEB. In fact, WUE
is given much attention as a physiological trait related to plant drought
resistance. Especially, the molecular research regarding the enhancement of
WUE playing a crucial part in the selection and cultivation of drought-resistant
or drought-tolerant crop varieties. When breeding for drought tolerance, biomass
productivity and WUE are considered as fundamental agronomic characteristics
(Blum, 1993).
WUE is the ability of the crop to produce biomass per unit of water transpired
and HI is the fraction of total dry matter harvested as yield (Liang et al., 2006).
The efficiency for producing dry matter per unit absorbed water, and the ability
to allocate an increased proportion of the biomass into grains (Manivannan et
al., 2007). In this scenario, WUE, TE and HI can be regarded as important
adaptive traits to drought environment (Sankar et al., 2008). In addition, Plant
WUE is an important index for measuring plant drought-resistant and yield
(Song et al., 2008). Water scarcity is a major factor limiting agricultural
production all over the world (Liu et al., 2005; Shao, et al., 2008). With the
change of global environment, the impact of drought stress on crop yield
becomes more serious (Song et al., 2008). Thus, for sustainable agricultural
development, irrigation practices must be implemented on the basis of applying
Chapter 4 Discussion
196
the available water resource more efficiently. WUE can be observed at several
levels. In recent years, numerous progresses have been made in the investigation
of plant WUE, especially at the molecular level (Shao et al., 2008).
In fact, unfavorable environmental factors lead to sharp changes in the
balance of growth hormones associated with not, only the accumulation of
ABA, but also with a decline in the level of the growth activating hormones
IAA, GA3 and cytokinins (Jackson, 1997). These changes would result in a new
endogenous hormone balance that would be favorable to the plant’s response to
drought. In the present investigation the water stress caused marked increases in
ABA levels in the flag leaves of two wheat cultivars and this was pronounced
with the sensitive one (Table13 and figure 31a). In addition, such accumulation
of ABA may be the reason of stomatal closure on both upper and lower sides of
the flag leaves. These results were in agreement with those obtained by
Assmann et al. (2000) and Raphael et al. (2003). Whether, the stomatal closure
system in plant leaves is abscisic acid (ABA)-dependent or -independent is
unknown (Rao et al., 2006) but, stomatal closure in many plants is incomplete
even after application of high concentrations of ABA (Mustilli et al., 2002).
However, field-grown plants, woody plants and wild watermelon plants show
almost complete stomatal closure and transpiration rates of almost zero during
severe drought stress (Yokota et al., 2002).
During vegetative growth, ABA-mediated adaptive responses are critical to
plant survival during drought, salt and cold stress (Dorothea and Ramanjulu,
2005). These stressors serve as a trigger for the accumulation of ABA, which in
turn activates various stress-associated genes that are thought to function in the
accumulation of osmoprotectants, (LEA) proteins, and signaling transcriptional
regulation.
Chapter 4 Discussion
197
Growth reduction under drought stress is mainly caused by a decrease in the
concentration of IAA (Saugy and River 1988). Nevertheless, there are reports
that there were no significant changes in IAA under drought stress (Li et al.,
2000) and that changes in IAA caused by drought have no significant regulatory
function in the process of adaptation of a plant to drought. However, relatively
little is known about the changes in plant endogenous GA3 under water stress
condition (Tian-hongl and Shao-hua, 2007). In this study, water stress showed
some inhibitory effect on endogenous GA3 in wheat flag leaves (Table13and
figure 31c). It is consistent with the results obtained in corn (Shi et al. 1994) and
in apple leaves (Tian-hongl and Shao-hua, 2007).
Water stress caused significant reduction in the cytokinins levels of the two
wheat cultivars (Table13and figure 32). The protective role of cytokinins is due
to their regulatory effects on the renewal of disrupted cellular structures, the
condition of the stomata, and de novo synthesis and activation of proteins that
are required for increasing plant resistance to water stress (Chernyadèv, 2005).
This is may probably be due to water stress restricts the transpiration rate by
inducing stomatal closure and thus inhibit the rate of translocation of CKs from
root toward shoot. Furthermore, water stress decreased the rate of biosynthesis
of CKs within the root system of wheat plants (Blackman and Davies, 1985). In
addition, many studies have investigated the role played by cytokinin in the
communication process among root, stem, and leaf under water stress (Kaur et
al., 2000).
This study showed that under water stress, ABA, IAA, GA3, zeatin, kinetin,
benzyl adenine concentration in flag leaves was significantly correlated with
RWC, SWD, stomatal area, transpiration rate, WUEG, WUEB, and consequently
grain yield indicating that growth hormones were involved in the plant water
relationships and its productivity (Tables 14&15).
Chapter 4 Discussion
198
Exogenous spray with GB, presowing treatment with SA or their interaction
may increase the drought tolerance of sensitive cultivar by acceleration of
growth promoters (IAA, GA3 and CKs) and at the same time reduced
accumulation of inhibitor represented by ABA in droughted plants. These results
were in accord with those obtained by Sakhabutdinova et al. (2003) and
Shakirova et al., (2003) who stated that, SA prevented the drought-induced
decline in concentration of IAA and cytokinins in seedlings of wheat plants
under sanity and reduced the accumulation of ABA. Maintaining a high level of
ABA under stress conditions in plants pretreated with SA is important as ABA
could play a regulating role in SA-induced unspecific plant resistance.
4.4. Osmolytes in Relation to Osmotic Adjustment
In order to understand the physiological adaptation of Triticum aestivum to
stress induced by water stress, osmotic pressure, proline, TSN, TSS, organic
acids and ions content in flag leaf sap were studied particularly after ear
emergence (at heading) and at the beginning of the grain set (at anthesis). Thus,
the results showed that water stress induced a marked increase in osmotic
pressure (Table 16 & Fig. 33a). This is probably due to the increases in proline,
TSN, TSS, organic acids and ions content. Furthermore, osmotic pressure
appeared to be positively correlated with organic osmolytes as well as inorganic
acids. In accordance to these results, Blum (1996) concluded that osmolytes
accumulation in plant cells results in a decrease of the cell osmotic potential and
thus in maintenance of water absorption and cell turgor pressure, which might
contribute to sustain physiological processes, such as stomatal opening,
photosynthesis, and growth expansion. Furthermore, occurrence of osmolytes
accumulation at sensitive crop reproductive stages has been reported to play a
constructive role against floral abortion (Wright et al., 1983), which results in
Chapter 4 Discussion
199
maintaining grain number under water deficit (Moinuddin and Khanna-Chopra,
2004). Additionally, osmolytes accumulation has also been claimed to facilitate
a better translocation of preanthesis carbohydrate reserves to the grain during the
grain-filling period (Subbarao et al., 2000).
Solutes accumulation in plant cells creates an intracellular osmotic potential
which in the presence of rigid cell wall generates turgor pressure. Maintaince of
turgor is necessary for maintance of growth through cell elongation (Yancey et
al., 1982). Moreover, the accumulation of these solutes may not be important for
osmotic stress tolerance but the metabolic pathways may have adaptive value
(Hasegawa et al., 2000). A further hypothesis is that compatible solutes are also
involved in scavenging reactive oxygen species (Chen and Murata, 2002).
The present results indicated that GB, SA or their interaction increased the
measured osmotic pressure and osmolytes concentration ( proline, total soluble
nitrogen, total soluble sugars, ions content (Na+, K
+, Mg
+2, Ca
+2 and Cl
- ) as well
as Na+/K
+in wheat plants subjected to water deficit. In fact, GB added more
increase to wheat osmotic pressure and these results are similar to those obtained
by Ibrahim (2004) with sorghum plants grown under salinity stress. In this
respect, Iqbal et al. (2008) found that water stress significantly decreased leaf
water contents, osmotic and turgor potentials in two sunflower lines and foliar
application of GB at the vegetative or reproductive growth stage increased leaf
water and turgor potentials to some extent in both sunflower lines when grown
under water stress. Moreover, Sulian et al. (2007) suggested that GB may not
only protect the integrity of the cell membrane from drought stress damage, but
also be involved in OA in transgenic cotton plants. Salicylic acid caused an
additional increase in wheat osmotic pressure as well as the studied osmolytes
and these were the same results obtained by Chinnusamy and Zhu (2003).
Chapter 4 Discussion
200
Proline content of the flag leaves increased at both stages of growth in both
wheat cultivars under water stress condition (Table 16 & Fig. 33b). The
accumulation of proline, primarily in the cytosol, often occurs in plants under
stress with strong correlation between stress tolerance and proline accumulation,
but the relationship is not universal and may be species dependent (Ashraf and
Foolad, 2007). There are other roles which proposed for proline besides osmotic
adjustment in stressed plants include acting as hydroxyl scavenger, stabilization
of membrane and protein structure, as a sink of carbon and nitrogen for stress
recovery, and buffering cellular redox potential under stress (Lee et al., 2008).
Moreover, high levels of proline enabled the plant to maintain low water
potentials (Sankar et al., 2007). By lowering water potentials, the accumulation
of compatible osmolytes, involved in osmoregulation allows additional water to
be taken up from the environment, thus buffering the immediate effect of water
shortages within the organism (Kumar et al., 2003).
It is important to note that proline accumulation with GB application in the
two wheat cultivars under water stress compared to drought-stressed plants
alone suggests that proline accumulation together with GB in leaves presumably
decreased the extent of drought-induced damage even more than drought-
stressed group alone. The protective role of GB was more pronounced in the
sensitive cv. Therefore, exogenous GB application might be a useful method to
improve growth and productivity of higher plants and retard aging process under
drought-stressed conditions.
An increase in proline concentration in SA-treated plants under normal and
stress conditions was also observed. Proline can thus be considered as an
important component in the spectra of SA-induced ABA-mediated protective
reactions of wheat plants in response to drought, contributing to a reduction in
the injurious effects of drought and an acceleration of the reparation processes
Chapter 4 Discussion
201
following stress, evidencing the protective action of SA on wheat plants
(Shakirova et al., 2003). .
In the present investigation the TSN accumulated in response to water stress
conditions in both wheat cultivars (Table 16 & Fig. 34a). Such accumulation
may be mainly resulted from a sharp increase in total free amino acids, total
soluble proteins and glycine betaine (Ibrahim, 2004). This change in nitrogen
content may be related to the inhibition of translocation from root to shoot,
inhibition of protein synthesis or the increase in protease activity (Khalil and
Mandurah, 1990). The increase in the soluble nitrogen compounds are of
importance in plant osmoregulation in response to water deficit. The application
of GB, SA or their interaction caused an accumulation in TSN in both wheat
cultivars at heading and anthesis.
Our findings indicate that water stress increased TSS in both wheat cultivars
(Table 16 & Fig. 34 b). This may result from increased starch hydrolysis,
synthesis by other pathways or decreased conversion to other products. Several
studies have shown an increase in amylase activity in water-stressed leaves (e.g.
Keller and Ludlow, 1993). Alternatively, increased translocation of
carbohydrates into leaves or a decrease in translocation of carbohydrates from
leaves could also contribute to the observed sugar accumulation. In this respect
Xue et al. (2008) found that water deficit in wheat leaves caused a reduction in
photosynthesis and high demands for osmolyte synthesis especially total soluble
sugars.
The marked increase in the content of soluble sugars in flag leaf sap of
stressed and nonstressed plants treated with GB, SA or their interaction agrees
with the results obtained by El Tayeb (2005b) who recorded an additional
increase in Na, soluble proteins and soluble sugars in salt-stressed barley grains
Chapter 4 Discussion
202
due to application of SA. SA treatments increased K+/Na
+ ratio in the plant
leaves under drought condition.
Organic acids content (citric acid and keto-acids) were increased in both
wheat cultivars after withholding water (Table 16 & Fig. 35). The increase in
organic acids content may be a result of drought induced synthesis (Venekamp
et al., 1989) and it is important for plant osmotic adjustment under water stress
(Morgan, 1984), and regulation of pH of plant cells (Venekamp et al.,1989).
These results were in a good conformity with those obtained by many others
who recorded the increase in organic acids in response to water stress (Ibrahim,
1999) and salinity (Shi and Sheng, 2005). Also, Hasaneen et al., (1990) found
that - ketoglutaric and some carboxylic acids of Krebs cycle increased in Zea
mays seedlings and plants in response to salinity stress.
The application of GB, SA or their interaction caused an accumulation in
organic acids in both wheat cultivars at heading and anthesis. The dramatic
increase in the organic acids in response to GB, SA or their interaction may
probably be due to the importance of organic acids in plants osmotic adjustment
under water stress (Morgan, 1984). Moreover, increased concentration of
organic acids may involve in oxidative respiration originate from enhanced
synthesis induced by dehydration and are directly linked to the proline synthesis
pathway, a mechanism which control the cytoplasmic pH level (Venekamp et
al., 1989).
An important results of the present study is that water stress, in the absence
of salinity in the root zone, induced a conspicuous increase in the flag leaves
ions (Na+, K
+, Mg
+2, Ca
+2 and Cl
- ) as well as Na
+/K
+ ratio (Table 17 & Fig. 36,
37). The effects of both stresses (water and salt stress) are not strictly additive in
reducing plant performance and that tolerances to water and salt stress are linked
Chapter 4 Discussion
203
through a common mechanism of Na uptake for osmotic adjustment (Glenn and
Brown, 1998). A plant in drying soils is exposed to increasing levels of both
water deficit and osmotic stress because the soil matrix potential decreases
simultaneously with decreasing soil moisture. Even if osmotic adjustment
occurs, a decrease in the hydraulic conductivity of root membrane is observed
and it has been clearly demonstrated that the impact of Na+ on water-channel
function is not due to its osmotic effect (Carvajal et al., 2000).
Another role of Na in C3 species such as wheat is related to its involvement
in photosynthesis (Brownell and Bielig, 1996). They also stated that, an in-
creased sodium concentration in plants experiencing water stress may be related
to an increase in the metabolic requirement of sodium to sustain photosynthesis
in these conditions. Sodium has been reported to be involved in the maintenance
of mesophyll chloroplast structure, mainly in relation to granal stacking and thus
sodium deficient C3 plants exhibit a wide range of chlorophyll a fluorescence
perturbations (Grof et al., 1986). Sodium may also be involved in the
regeneration of phosphoenolpyruvate in mesophyll chloroplasts (Murata et al.
1992) because a Na+ gradient across the envelope could be an alternative energy
source for the active transport of pyruvate (Ohnishi et al., 1990). An increase in
sodium uptake could then be the consequence of a stress-induced decrease in the
efficiency of the Na+/pyruvate co transport-system. This confirms the previous
findings of Martınez et al. (2003) who reported that drought-induced specific
increase in Na+ concentration in two populations of Atriplex halimus under
water stress.
The application of GB, SA or their interaction caused an accumulation in Na+ in
both wheat cultivars at heading and anthesis. This is in agreement with Ibrahim
(2004) who studied the efficacy of exogenous glycine betaine application on
sorghum plants grown under salinity stress and found that salinity reduced
Chapter 4 Discussion
204
sorghum growth. Foliar application of glycine betaine at 75 mM mitigated the
adverse effects of salinity. The measured osmotic pressure and solutes
concentration (Na, K, Ca, Mg, total soluble sugar and betaine) increased while
K/Na ratio decreased in sorghum plants. Glycine betaine added more increase to
sorghum osmotic pressure and increased the concentration of soluble sugars
Potassium content was increased to some extent in both wheat cultivars at
heading and anthesis, higher K+ concentrations, effectively stabilize native
protein (Borowitzka, 1981).This had been observed by (Ibrahim, 1999).
Moreover, in this investigation the drought resistant cultivar had higher K+
concentrations in the face of drought stress more than the sensitive variety and
this reflects the important role of this cation in wheat plants adaptation to water
stress conditions.
The application of GB, SA or their interaction caused an accumulation in K+
in both wheat cultivars at heading and anthesis. In this respect, Günes et al.
(2005) demonstrated that SA treatments caused N accumulation in plants and
increased P, K, Mg and Mn concentrations under stress conditions. On the other
hand, Hamada and Al-Hakimi (2001) observed positive effects of SA in the Na,
K, Ca and Mg content of wheat plants grown under salinity. SA application
inhibited Na accumulation in salinity condition. Thus seed pretreatment with SA
induced a reduction in sodium absorption and toxicity, which was further
reflected in low membranes injury, high water content and dry matter
production.
Our data showed that, calcium ion accumulated in response to water stress.
Ca2+
plays important roles in plant responses to drought resistance. Nayyar
(2003) also found in wheat that Ca2+
appeared to reduce the devastating effects
of stress by elevating the content of proline and glycine betaine, thus improving
Chapter 4 Discussion
205
the water status and growth of seedlings and minimizing the injury to
membranes. In fact, Calcium ion has unique properties and universal ability to
transmit diverse signals that trigger primary physiological actions in cells in
response to hormones, pathogens, light, gravity, and stress factors. Being a
second messenger of paramount significance, calcium is required at almost all
stages of plant growth and development, playing a fundamental role in
regulating polar growth of cells and tissues and participating in plant adaptation
to various stress factors. Many researches showed that calcium signals decoding
elements are involved in ABA-induced stomatal closure and plant adaptation to
drought, cold, salt and other abiotic stresses and some new studies show that
Ca2+
is dissolved in water in the apoplast and transported primarily from root to
shoot through the transpiration stream (Song et al., 2008).
Glycine betaine, salicylic acid or their interaction treatments added more
increases in Ca2+
content in wheat cultivars. These results were in a good
agreement with Raza et al. (2007) who reported higher increases in GB, K+,
Ca2+
and K/Na ratio in two cultivars of wheat (tolerance and salt sensitive)
treated with GB under salt stress. High accumulation of GB and K+ mainly
contributed to osmotic adjustment, which is one of the factors known to be
responsible for improving growth and yield under salt stress.
Magnesium content was increased to some extent in both wheat varieties at
heading and anthesis, this increase was also demonstrated by Ibrahim (1999)
who reported that drought stress favors the accumulation of Mg+2
in plants. On
the other hand the applied chemicals added more increase in the Mg+2
of wheat
cultivars. These are the same result obtained by Ibrahim (2004).
4.5. Yield Components and Biochemical Aspects of Yielded Grains:
Chapter 4 Discussion
206
Yield is a result of the integration of metabolic reactions in plants;
consequently any factor that influences this metabolic activity at any period of
plant growth can affect the yield (Ibrahim and Aldesuquy, 2003). This is a well
established fact that yield of crop plants in drying soil reduces even in tolerant
lines of that crop species (Sankar et al., 2008; Shao et al., 2008). Yield and
yield attributes (shoot length, spike length, plant height, main spike weight,
number of spikelets per main spike, 100 kernel weight, grain number per spike,
grain weight per plant, straw weight per plant, crop yield per plant, harvest,
mobilization and crop index) were reduced due to water stress in both wheat
cultivars (table 19 and figures 38, 39.40, 41 &42). The reduction in yield of
stressed wheat plants was attributed to the decrease in photosynthetic pigments,
carbohydrates accumulation (polysaccharides), nitrogenous compounds (total
nitrogen and protein). The decrease in yield and yield components in different
crops has also been reported by many workers (Tahir et al., 2002; Arfan et al.,
2007; Sankar et al., 2008). These workers clearly indicated that drought tolerant
genotypes showed less reduction in yield plants in respect of susceptible ones.
Hence, maintainenance of better yield of the wheat cultivar, Sakha 93 resistan
than that of Sakha 94 under water deficit.
Drought stress during the early stage of reproductive growth tends to reduce
yield by reducing seed number. Stress later in the reproductive period during
seed development reduces yield primarily from reduced seed size. Prolonged
moisture stress during reproductive growth can severely reduce yield because of
reduced seed number and seed size (Dombos et al., 1989). Furthermore, water
stress reduced the head diameter, 100-achene weight and yield per plant in sun
flower (Blumwald et al., 2004; Shao et al., 2008). These authors also observed
significant but negative correlation of head diameter with fresh root and shoot
weight under water stress. A positive and significant relation was recorded
Chapter 4 Discussion
207
between dry shoot weight and achene yield per plant. The yield components,
like grain yield, grain number, grain size, and floret number, were decreased
under pre-anthesis drought stress treatment in sunflower (Shao et al., 2008).
Zhang et al. (1998) found from field-grown wheat that a soil drying during
the grain filling period could enhance early senescence. They found that while
the grain filling period was shortened by 10 days (from 41 to 31 days) in
unwatered (during this period) plots, a faster rate of grain-filling and enhanced
mobilization of stored carbohydrate minimized the effect on yield. They showed
that the early senescence induced by water deficit does not necessarily reduce
grain yield, even when plants are grown under normal nitrogen conditions. The
gain from accelerated grain-filling rate and improved translocation outweighed
the possible loss of photosynthesis as a result of shortened grain filling period
when subjected to water stress during grain filling. Monocarpic plants such as
wheat and rice need the initiation of whole plant senescence so that stored
carbohydrates in stems and leaf sheaths can be re-mobilized and transferred to
their grains (Ali et al., 2007). Delayed whole plant senescence leads to poorly
filled grains and unused carbohydrate in straws (Zhang and Yang, 2004b). Slow
grain filling may often be associated with delayed whole plant senescence (Ali
et al., 2007).
Water stress reduced harvest, mobilization and crop index in the two wheat
cultivars (Table 19 & Fig. 24). This was in agreement with Jaleel et al. (2008)
who reported that, water stress decreased harvest index, and biomass yield in
two varieties of Catharanthus roseus (L.). However, in crops, the detrimental
effects of water deficits on the harvest index (HI) also minimize the impact of
the water limitation on crop productivity and increase the efficiency of water use
(Chaitanya et al., 2003). Therefore, increasing transpiration, transpiration
Chapter 4 Discussion
208
efficiency and harvest index are three important avenues for the important of
agricultural productivity (Sankar et al., 2008). Additionally, the aerial
environment plays a role in determining the ratio of carbon gain to water use,
because the vapor pressure deficits between the leaf and the air determine the
transpiration rate (Zhu et al., 2002).
The results clearly indicated that application of GB (10 mM) was significant
in alleviating the adverse effects of water deficit on yield and yield components
of both wheat cultivars. These results were in conformity with those obtained by
Ibrahim and Aldesuquy (2003) with sorghum plant under drought stress. This
improvement in yield and yield components due to GB application would results
from the beneficial effect of GB on growth and metabolism and its role as
osmoprotectant. However, there are however, some contrasting reports
indicating no effect of supplied GB on yield of wheat (Agboma et al., 1997a)
and cotton (Naidu et al., 1998; Meek, et al., 2003). In this respect, Iqbal et al.
(2005) recorded that, exogenous supply of the GB (foliar spray) showed
effective role in ameliorating the effects of water stress on turgor potential and
yield of two sunflower lines. The effect of GB application was more pronounced
when it was applied at the time of initiation of water deficit at the vegetative or
reproductive growth stages.
The positive effects of foliar spray of GB on 100- kernel weight of wheat
cultivars grown under water stress was in accord with the results observed by
some investigators in different crops [sunflower (Iqbal et al.. 2008); tomato
(Makela et al., 1998), tobacco (Agboma et al., 1997b), maize (Agboma et al.,
1997a), cotton (Gorham et al., 2000) and wheat (Diaz-Zarita et al., 2001)].
The application of salicylic acid (0.05 M) enhanced the yield and yield
components of the two wheat cultivars. In this respect, Arfan et al. (2007)
Chapter 4 Discussion
209
studied the effect of exogenous application of salicylic acid (SA) through the
rooting medium of two wheat cultivars differing in salinity tolerance. They
found that increase in grain yield along with increase in 100-grain weight,
number of grains and number of spikelets per spike with 0.25mM SA
application under saline conditions suggested that improvement in salt-induced
reduction in grain yield with SA application was mainly due to increase in grain
size and number.
It can be stated that the beneficial effect of SA on grain yield may have been
due to translocation of more photoassimilates to grains during grain filling,
thereby increasing grain weight. These results are similar to those of Zhou et al.
(1999) who reported that maize plants stem injected with SA produced 9% more
grain weight than those with sucrose and distilled water treatments. The second
possible mechanism of SA-induced yield enhancement might be an increase in
the number of spikelets and number of grains, because SA has the capacity to
both directly or indirectly regulate yield. These results were in a good
agreements with those obtained by (Khan et al., 2003) with maize, and (Kumar
et al., 2000; Khan et al., 2003) with soybean. In addition, boll number in cotton
was found to be up-regulated by SA application (Hampton and Oosterhuis,
1990).
5.5.2. Grain Biomass and its Biochemical Aspects:
Generally, the grain fresh and dry masses, carbohydrates and total protein
were decreased in response to water stress in both two wheat cultivars (Tables
20 and Fig. 43, 44&45). The results showed that, water withholding occurred
during grain filling particularly the 2nd stress period (at anthesis) and this firstly,
lead to increase in ABA levels in flag leaves which induced stomatal closure and
consequently decreased photosynthetic activity in flag leaves (the main source
Chapter 4 Discussion
210
of photo-assimilates towards developing grains). The pervious effect of water
stress may result in a decrease in the grain biomass. Secondly, water stress
decreased the leaf area by inducing leaf rolling particularly in susceptible
cultivar and this may decrease the dry matter production which translocated
towards developing grains. Thirdly, water stress may stimulate the early
senescence in wheat leaves particularly in susceptible cultivar which also
affected the translocation the photo-assimilates from leaves (particularly flag
leaf) which represents the main export source towards the main import source
(developing grain). Bearing in conclusion of Egeli et al (1985) that the
accumulation of dry matter by grains requires the production of assimilates in
the leaves, their translocation to the fruit, movement into the storage organs of
seed, and the synthesis of materials to be stored.
The above maintained results were in accord with those obtained by many
investigators (Sankar et al. 2007). In addition, Savin and Nicolas (1996)
investigated the effects of drought stress on grain growth, starch and nitrogen
accumulation in barley cultivars. Water deficits decreased both individual grain
weight and grain yield. Nitrogen content per grain was quite high and similar for
all treatments, and nitrogen percentage increased when stress was severe enough
to reduce starch accumulation. This confirms that starch accumulation is more
sensitive to post-anthesis stress than nitrogen accumulation.
Application of GB, SA or their interaction appeared to mitigate the
deleterious effects of water stress on grain biomass of the two wheat cultivars.
The repairing effect of SA may be attributed to the fact that SA reduces the rate
of transpiration from leaves (Larque-Saavedra, 1979), which could possibly lead
to the accumulation of excessive water, thus resulting consequently in an
increase in grain fresh mass (Abo-Hamed et al., 1990). Furthermore, GB
application may act in the same manner as SA in inducing drastic reduction in
Chapter 4 Discussion
211
the rate of transpiration. The results obtained from diurnal changes in
transpiration rate make this postulation decisive (see 3.4).
The results in table 20 and figure 44&45 indicated that, soluble sugars were
accumulated in response to water stress in both wheat cultivars. On the other
hand, water stress induced massive decrease in polysaccharides content in
yielded grains of both wheat cultivars. This may probably due to water stress
stimulates the degradation of polysaccharides and at the same time increase of
dark respiration during which apart of soluble sugars were consumed as a
respiratory substrate. The other part of soluble sugars may explain the massive
increase in total soluble sugars occurred within the developing grain as result of
water stress. From anther point of view, water stress decreased the pigments
formation in wheat leaves resulting in inhibition in photosynthetic activity which
in turn lead to less accumulation of carbohydrates in developed leaves and
consequently may decrease the rate of transport of carbohydrates from leaves to
the developing grains, where there is a good relationship between source
(leaves) and sink (grain) in cereal plants. Furthermore, the noticed decrease in
polysaccharides of wheat grains as a result of water stress could explained on
the fact that, water stress impaired the utilization of carbohydrates during the
vegetative growth and reduced the area of conductive canals (mainly phloem
and xylem), so reduction in the translocation of the assimilates toward the
developed grains may be occurred.
In accord to these results, several physiological studies suggested that under
stress conditions nonstructural carbohydrates (sucrose, hexoses, and sugar
alcohols) accumulate although to varying degree in different plant species. A
strong correlation between sugar accumulations and osmotic stress tolerance has
been widely reported, including transgenic experiments (Streeter et al., 2001;
Taji et al., 2002). In addition Singh et al. (2008) reported that the effect of water
Chapter 4 Discussion
212
stress (WS) at 8 and 15 days post anthesis (DPA) on the characteristics of starch
and protein separated from five wheat varieties. WS-induced changes in A-, B-
and C-type granules distribution were variety- and stage-dependent. The starch
from wheat exposed to WS at 15 DPA showed lower amylose content, lipids
content and pasting temperature, and higher peak viscosity, final viscosity and
setback.
The data in table 20 & figure 46 indicated that phosphorus content (organic,
inorganic and total) in wheat grains increased due to water stress application.
Several studies have investigated the relationship between phosphorus status and
photosynthetic metabolism (Rao and Terry, 1994) but few have evaluated the
effects of water deficit in this relationship (Dos Santos et al., 2006). El-Taybe
(2005b) recorded that phosphorus increased in barley plants due to salinity. In
addition, application of GB, SA or their interaction caused additional
accumulation in phosphorus content in both wheat cultivars yielded grains.
Water stress stimulates the accumulation of both calcium and sodium content
but decreased the magnesium and chloride content in the yielded grains of the
two wheat cultivars (Table 20 and Fig. 47). This increase in calcium and sodium
levels may result from transportation of these elements from root to shoot
through the transpiration stream till reach to developing grains. In addition,
application of GB, SA or interaction seemed to induce additional increase in
ionic content (calcium, sodium, magnesium and chloride) of the developed
grains.
Under water stress, protein content of the developed grains was significantly
decreased in both wheat cultivars (Table 20 & Fig. 48). The decrease in protein
contents in yielded grains was more pronounced in the sensitive cultivar than the
resistant ones under drought, this may probably be due to less transport of
Chapter 4 Discussion
213
protein from source (flag leaf) to the sink (grain). In support of this finding in
this study, water stress induced remarkable decrease in soluble protein in flag
leaf at heading and anthesis.
The decreased in protein content in yielded grains as a result of drought stress
was alleviated by the application of GB, SA or their interaction. In connection
with these results, Mäetak el al. (2000) found increased protein in tomato plants
under drought or salinity by means of foliar-applied GB. In addition, similar
results were obtained by El-Taybe (2005b).
Free amino acids play an important role in maintaining the osmotic balance in
the tissue of plants, yeasts, bacteria and animals (Zushi et al., 2005). In this
investigation, water stress induced a massive increase in total amino acids
detected in the harvested grains of the two wheat cultivars (Table 21&22). This
may result from the enhanced production of amino acids as results of increased
proteolytic activities which may occur in response to the changes in osmotic
adjustment of their cellular contents (Greenway and Munns, 1980). The
accumulation of free amino acids under stress at all the growth stages indicates
the possibility of their involvement in osmotic adjustment (Yadav et al., 2005).
Osmotic adjustment is one of the important mechanisms alleviating some of the
detrimental effects of water stress (Morgan, 1984).
The amino acid content has been shown to increase under drought conditions
in coconut (Kasturi Bai and Rajagopal, 2000), in wheat (Hamada, 2000), in
Arachis hypogaea (Asha and Rao, 2002), in sorghum (Yadav et al., 2005), in
pepper (Nath et al., 2005), in Abelmoschus esculentus (Sankar et al., 2007).
The obtained results indicated that glutamic acid was the most abundant
amino acid in the yielded grains of the control and treated plants. These results
were in a good agreement with those obtained by Winter et al. (1992); Caputo
and Barneix (1997). They found that, the amino acid composition of phloem sap
Chapter 4 Discussion
214
is different in different species, in barley, Glu accounts for approximately 50%
of the total amino acids, while Asp accounts for roughly 20% and in wheat, Glu
amounted to 30% of the total amino acids, and Asp to 20%, with these
proportions changing with plant age. Also, in spinach, Glu was the most
abundant amino acid, accounting for 39.1%, followed by Asp (14.7%) and Gln
(10.1%) (Riens et al., 1991), and Glu was also the dominating amino acid in the
phloem of Beta vulgaris L. (Lohaus et al., 1994).
For instance, an accumulation of Glu has been reported in the wheat grains
under salinity (Aldesuquy, 1998); roots and leaf blades of barley (Yamaya and
Matsumoto, 1989) while, in Phragmites australis, Glu levels rose in rhizomes
(Hartzendorf and Rolletschek, 2001). In strawberry fruit, salt stress led to a
considerable increase in Glu, especially in the salt-sensitive cv. Elsanta
(Keutgen and Pawelzik, 2008). This accumulation of Glu could have been
caused by both, the activation of biosynthesis from Glu and the inactivation of
Glu degradation (Yoshiba et al., 1997).
Water stress induced an accumulation in proline concentration in harvested
grains in both wheat cultivars (Table 21&22). Increased proline in the grain of
stressed wheat plants may be an adaptor to overcome the stress conditions.
Proline accumulates under stressed conditions supplies energy for growth and
survival and thereby helps the plant to tolerate stress (Chandrashekar and
Sandhyarani, 1996). Similar results were obtained in Gossypium hirsutum
(Ronde et al. 1999), in wheat (Hamada, 2000), in sorghum (Yadav et al., 2005),
in bell pepper (Nath et al., 2005), and in salt-stressed Catharanthus roseus
(Jaleel et al., 2007).
Treatments with GB, SA or their interaction induced remarkably increases in
amino acids detected in the harvested grains of both wheat cultivars. This was in
agreement with El-Taybe (2006) with sunflower plants treated with SA under
Chapter 4 Discussion
215
Cu- stress conditions. Also, Hussein et al. (2007) studied the effect of salicylic
acid and salinity on growth of maize plants. They found that all amino acid
concentrations were lowered by salinity except for proline and glycine. All
determinate amino acid concentrations (except methionine) were increased with
the application of salicylic acid (200 ppm). On the other hand, methionine was
negatively responded which slightly lowered. These chemicals may reduce
proline oxidase and resulted in proline accumulation which acts as an osmolyte
as well as scavenger.
The applied chemicals appeared to mitigate the effect of water stress on
wheat yield particularly the sensitive one and the biochemical aspects of yielded
grains. The effect was more pronounced with glycine betaine + salicylic acid
treatment. This improvement would result from the beneficial effect of the
provided chemicals on growth and metabolism of wheat plants under water
deficit condition.
It is clear from this investigation that the effect of water stress on both wheat
cultivars particularly sensitive one had a negative effect on growth, metabolism
and productivity. On the other hand, the exogenous application of GB, SA or
their interaction displayed a positive role in improving the growth vigour of both
root and shoot as well as the ability of both wheat cultivars (particularly the
sensitive one) to overcome water deficit conditions by increasing the following:
1. Hormonal regulation and improvement of leaf turgidity by causing stomatal
closure, decreasing the rate of transpiration, increasing relative water content
as well as water use effeinicy for economic yield.
2. Accumulation of osmolytes [proline, total soluble nitrogen, total soluble
sugars, organic acids, ions (Na+, K
+,Ca
+2, Mg
+2 and Cl
-) content as well as
Na+/K
+ ratio in cell sap of flag leaves)] which play a major role in osmotic
adjustment.
Chapter 4 Discussion
216
3. Stimulate leaf expansion, enhancing the production of photosynthetic
pigments and inducing modification in conductive canals particularly phloem
which participates in translocation of photo-assimilates from flag leaves
towards developing grains (where there was a good correlation between
phloem area and grain yield). Therefore the applied chemicals particulary
GB+SA increase the yield capacity of the wheat plants.
