Effects of Three Different Flooding Periods on Some Anatomical, Morphological and Biochemical Changings in Maize (Zea mays L.) Seedlings
Flooding stress has many important morphological
and biochemical effects on plants. Because of the importance of determination
the effects of flooding on the plants and understanding of the tolerance
mechanisms, in this research Four-days-old maize (Zea mays L.)
seedlings (cv. single cross 704) were exposed to 4, 7 and 10 days flooding
stress. At the end of each treatment the roots and shoots of the seedlings
were harvested separately. To show the some anatomical, morphological
and biochemical changings of the flooding on plants, cross sections of
the roots and shoots were studied with light microscope. There was no
clear changing in the tissue structures` of leaves and stems of different
treatments in comparison with controls, but in the roots of plants aerenchyma
had been developed under stress condition especially in the mesocotyl
region. The roots of flooded plants grow towards the soil surface despite
positive geotropism of control roots. The chlorophyll a and b content
and the ratio of chlorophylls a/b have been decreased but the amounts
of soluble sugars have been increased in both the roots and shoots of
seedlings. We conclude that flooding influences plants growth and life
and development of the aerenchyma and vegetative roots help to plants
to adapt itself to stress condition. So it is very important to know which
plants are sensitive or tolerant and what are the tolerance mechanisms
in the different plants to succeed in agricultural efforts.
Flooding occurs when all of the pores of the soil are
saturated with water and the amount of soil gases as oxygen have reduced
highly because the diffusion of O2 in water is 10,000 times
lower than the air (Ram et al., 2002). Plants require a free exchange
of atmospheric gases through the soil for their natural root growth and
metabolism. Reduction of the oxygen below the optimum levels (Hypoxia)
is the most common form of stress in wet soils that causes the roots of
plants submerge in water while the shoots are out in the atmosphere (Ram
et al., 2002). In such condition, because of the low oxygen in
the soil, respiration rate of the roots reduces and the plants maintain
their energy from fermentative metabolism in the roots to the survive
(Liao and Lin, 2001; Mohanty et al., 1993). On the other hand,
photosynthetic capacity has also been shown to be significantly inhibited
in flooding-intolerant plants (Liao and Lin, 2001). Adaptive mechanisms
to secure a renewed supply of oxygen to flooded root tissues include the
development of aerenchyma that allows the oxygen to move from the aerobic
shoots to the anaerobic roots. The aerenchyma differs in the origin among
species and it may be either lysigenous or schizogenous. Lysigenous aerenchyma
develops as a consequence of senescence of specific cells followed by
their autolysis and disintegration, whereas the schizogenous aerenchyma
develops by the cell separation and division (Igamberdiev et al.,
2005). In monocots such as maize and rice, the natural lysigenous aerenchyma
develops in the roots cortex toward the endodermis, above the root tip,
in regions where cells growth has been completed (Jackson, 1985). In the
Rumex species, a new aerenchymatous root system develops near the
soil surface in response to the flooding. This mechanism has supported
with oxygen concentrations for more than 50% of the total root respiration
under the Hypoxia conditions (Laan et al., 1990). The aim
of this research is to study the effects of three different flooding periods
on some anatomical, morphological and biochemical changings in Zea
mays L. seedlings.
MATERIALS AND METHODS
In this experiment, seeds of the Zea mays L. (cv.
Single cross 704) were obtained from the Agricultural Research Center
of Urmia during three mounts in 2007. The seeds are incubated in 25 °C
to germinate, after three days they were transferred to pots (diameter
15 cm) filled with the sterile vermiculate soil under the controlled condition
with 16/8 h light/dark photoperiod and 25/23 °C day/night temperature
(Hsu et al., 2000). After 24 h, the seedlings were irrigated for
4, 7 and 10 days. At the end of each treatment, the roots and shoots of
the seedlings were harvested separately. to show the effect of flooding
on the plants, the changes of the amounts of soluble sugars and chlorophylls
contents and the anatomical changes of the roots and shoots tissues were
studied. To prepare cross-sections of the shoots and primary roots, the
roots and shoots of all treatments and controls were taken and prepared
to do a paraffin-cut section for anatomic observation by the method of
Lin and Yeh (1996) and then they were studied by light microscope (Magnification
1000). Chlorophylls were extracted from the seedings using Lichentaller
method (Zhang and Kirkham, 1996). After extraction, chlorophyll (a) and
(b) in the youngest leaves were measured with spectrophotometrically (LKB
UV/Visible) at the 664 and 647 nm and their amounts were calculated using
following formula (Zhang and Kirkham, 1996).
Chla = 12/25A664-2/79A647
and Chlb = 21/51A647-5/10A664
||= Chlorophyll a
||= Chlorophyll b
||= Amount of absorption
Similarly, soluble sugars were determined with the phenol-sulfuric
method according to Fales (1979). For this, 0.5 g of the roots and shoots
were used and then it was filtered. Two milliliter from each sample was
taken; 1 mL 5% phenol was added then 5 mL 98% sulfuric acid was added
to the samples. After coolness and complete colour emergence of the solutions,
sugar contents were maintained by using spectrophotometer at 485 nm (Hsu
et al., 2000). All obtained data were analysed with Analysis of
Variance (ANOVA1), statistical software, than, compared the means by Duncan
test (Heim et al., 1990).
RESULTS AND DISCUSSION
Anatomical changes of flooding: The results of
tissues studies of the roots and shoots of plants exposed to different
periods of flooding indicated that there was no clear change in leaf and
stem tissues; while in the flooded roots aerenchyma has been developed
in comparison with controls (Fig. 1 ). The Aerenchyma
formation creates an internal gas exchange channel from the aerobic shoot
to the hypoxic roots. Air enters through stomata of leaves or lenticels
on the stem and passes through the network of aerenchyma channels to the
submerged roots. Oxygen consumption in the roots creates a negative pressure
gradient that draws air by mass flow to the roots, which in rice (Oryza
sativa) has been measured about 20 mL h-1 (Raskin and Kende,
1985). Another typical symptom of flooding in this research was the Hypertrophic
growth in the mesocotyl of 10 days flooded seedlings (Fig.
2). This type of growth that appears as a swelling at the area between
the base of the stems and roots are important because of the transitional
role of mesocotyl in carrying O2 from shoots to roots. The
reason for these changings depends on the radial cells division and their
expansion and is often accompanied by cell collapse and the aerenchyma
formation. It is consequently considered to be an adaptive mechanism that
causes increased air diffusion from shoots to roots (Visser and Voesenek,
2004). Another early symptom of flooding stress involves reorientation
of growth processes; for example, roots of flooded plants tend to become
negative geotropism. They grow upwards (Fig. 3) and
are able to receive more air from the soil surface (Jackson and Drew,
1984). The third changing is the development of adventitious roots in
flooded plants (Fig. 3). Flooding often causes malfunctioning
of roots formed prior to flooding, even in wetland species. This may eventually
lead to the death of a considerable part of the root system and a fast
replacement by well-adapted adventitious roots that contain more aerenchyma
than the original roots (Visser et al., 1996).
Changes of the chlorophylls contents: The amount
of chlorophyll a and b in the leaves of flooding treated plants was found
to be significantly lower than controls (Fig. 4). Reduction
of chlorophyll contents in hypoxia stress is probably due to the slowly
synthesis and fast destruction of chlorophyll pigment (Ashraf, 2003).
The ratio of chlorophyll b/a has also been decreased (Fig.
4). This reduction is because that the sensitivity of chlorophyll
b against flooding stress is more than chlorophyll a (Zaidi et al.,
2003). Previous studies suggest that chlorophyll b as a main part of photosystems
breaks down higher than chlorophyll a under the stress conditions (Mauchamp
and Methy, 2004).
The soluble sugars contents: Soluble sugars levels
in flooded roots and shoots have been increased more than controls (Fig.
5). A high level of fermentative metabolism in roots has been shown
to be important for plant survival because it supplies a high enough energy
charge that can sustain metabolism in the roots (Mohanty et al.,
1993). Thus, maintaining adequate levels of fermentable sugars in the
flooded roots is undoubtedly important for the long term survival of plants
during flooding. The changings of
|| Cross-sections of Zea mays seedling roots, (A)
the aerenchyma tissue developed in the flooded root seedlings for
10 days (x50) in comparison with controls (B). (C) the initial aerenchyma
tissue formation in flooded root seedlings flooded for 4 days (x50)
in comparison with controls (D)
|| Cross-sections of Zea mays seedlings mesocotyles,
(A) the aerenchyma tissue developed in flooded mesocotyle seedlings
for 10 days (x100) in comparison with controls (B). (C) abnormal mesochotyle
at 7 days after flooding (x100) in comparison with control (D)
||The roots of flooded Zea mays plants tend to
become negatively gravitropic and adventitious roots develop against
||The changings of total chlorophyll a and b content in
Zea mays L. seedlings exposed to different periods of flooding
(during 4, 7 and 10 days)
||The changings of total soluble sugars in Zea mays
L. seedlings roots and shoots exposed to different periods of flooding
(during 4, 7 and 10 days)
soluble sugars in roots of Zea mays during flooding
periods are agreed strongly with studies on alfalfa by Barta (1988) and
Castonguay et al. (1993) that reports the root starch can be mobilized
and converted to soluble sugar at the early stages of flooding. The starch
levels in roots of flooded corn were found to decrease markedly during
the early flooding stages (Su et al., 1998) may cause the soluble
sugars to increase. Analyses of soluble sugar in roots revealed that the
amounts of soluble sugar has increased 1.5-2 fold as compared controls
during the early stage of flooding, but by increasing flooding period
duration this ratio has decreased and the amount of sugars gradually decreased
and finally has reached to the levels similar of the controls (Fig.
5). Since consumption of soluble sugar under fermentative metabolism
during flooding condition has been increased (Mohanty et al., 1993)
and under Hypoxia, starch accumulation in the leaves has been attributed
to a reduced rate of translocation of carbohydrates from leaves to roots
(Barta, 1987), which apparently causes the carbohydrate demands to decrease
(Hsu et al., 1999).
There are vast flooded areas in the world and the plants
respond differently to flooding stress. So it is important to understand
how plants especially major crops such as Zea mays behave against
low external O2 concentration and adapts its growth and metabolism
over the short and long-term flooding stress. Moreover we founded that
the flooding stress causes some anatomical, morphological and biochemical
changes in plants; development of aerenchyma and adventitious roots are
more recessive factors that increases hypoxic tolerance in Zea mays.
Ashraf, M., 2003.
Relationships between leaf gas exchange characteristics and growth of differently adapted populations of Blue panicgrass (Panicum antidotale
Retz.) under salinity or waterlogging. Plants Sci., 165: 69-75.CrossRef | Direct Link |
Barta, A., 1987.
Supply and partitioning of assimilates to roots of Medicago sativa
L. and Lotus corniculatus
L. under anoxia. Plant Cell Environ., 10: 151-156.CrossRef | Direct Link |
Barta, A., 1988.
Response of field grown alfalfa to root waterlogging and shoot removal. I. Plant injury and carbohydrate and mineral content of roots. Agron. J., 88: 889-892.CrossRef |
Castonguay, Y., P. Nadeau and R. Simard, 1993.
Effects of flooding on carbohydrate and ABA levels in roots and shoots of alfalfa. Plant Cell Environ., 16: 695-702.CrossRef | Direct Link |
Fales, F.W., 1951.
The assimilation and degradation of carbohydrate by yeast cells. J. Biol. Chem., 193: 113-124.PubMed |
Heim, D.C., G.G. Kennedy and J.W. Vanduyn, 1990.
Survey of insecticide resistance among north carolina colorado potato beetle (Coleoptera: Chrysomelidae) populations. J. Econ. Entomol., 83: 1229-1235.CrossRef | Direct Link |
Hsu, Y.M., M.J. Tseng and C.H. Lin, 1999.
The fluctuation of carbohydrates and nitrogen compounds in flooded wax-apple trees. Bot. Bull. Acad. Sin., 40: 193-198.Direct Link |
Hsu, F.H., J.B. Lin and S.R. Chang, 2000.
Effects of waterlogging on seed germination, electric conductivity of seed leakage and developments of hypocotyl and radicle in sudangrass. Bot. Bull. Acad. Sin., 41: 267-273.Direct Link |
Igamberdiev, A.U., K. Baron, N. Little, M. Stoimenova and R.D. Hill, 2005.
The haemoglobin/nitric oxide cycle: Involvement in flooding stress and effects on hormone signaling. Ann. Bot. A Rev., 96: 557-564.Direct Link |
Jackson, M.B. and M.C. Drew, 1984.
Effects of Flooding on Growth and Metabolism of Herbaceous Plants. In: Flooding and Plant Growth, Kozlowski, T.T. (Ed.). Academic Press, Orlando, Florida, ISBN: 0-8243-0636-8, pp: 47-128
Jackson, M.B., 1985.
Ethylene and responses of plants to soil waterlogging and submergence. Annu. Rev. Plant Phys., 36: 145-174.Direct Link |
Laan, P., M. Tosserams, C.W.P.M. Blom and B.W. Veen, 1990.
Internal oxygen transport in Rumex
species and its significance for respiration under hypoxic conditions. Plant Soil, 122: 39-46.CrossRef | Direct Link |
Liao, C.T. and C.H. Lin, 2001.
Physiological adaptation of crop plants to flooding stress. Proc. Natl. Acad. Sci., 25: 148-157.Direct Link |
Lin, J.B. and M.S. Yeh, 1996.
Development of embryo and endosperm derived from selfing and inter specific hybridization between Glycine max
and G. tomentella
. J. Agric. Assoc. China, 173: 17-27.
Mauchamp, A. and M. Methy, 2004.
Submergence-induced damage of photosynthetic apparatus in Phragmites australis
. Environ. Exp. Bot., 51: 227-235.Direct Link |
Mohanty, B., P.M. Wilson and T. Rees, 1993.
Effects of anoxia on growth and carbohydrate metabolism in suspension cultures of soybean and rice. Phytochemistry, 34: 75-82.CrossRef | Direct Link |
Ram, P.C., B.B. Singh, A.K. Singh, P. Ram, P.N. Sing, H.P. Singh, J. Boamfa, F. Harren, E. Santosa, M.B. Jackson, T.L. Setter, J. Reuss, L.J. Wade, V.P. Singh and R.K. Singh, 2002.
Submergence tolerance in rainfed lowland rice: Physiological basis and prospects for cultivar improvement through marker aided breeding: A review. Field Crop Res., 76: 131-152.Direct Link |
Raskin, I. and H. Kende, 1985.
Mechanism of aeration in rice. Science, 228: 327-329.CrossRef | Direct Link |
Su, P.H., T.H. Wu and C.H. Lin, 1998.
Root sugar level in flooded luffa
and bitter melon is not referential to flooding tolerance. Bot. Bull. Acad. Sin., 39: 175-179.Direct Link |
Visser, E.J.W., C.W.P.M. Blom and L.A.C.J. Voesenek, 1996.
Flooding-induced adventitious rooting in Rumex
: Morphology and development in an ecological perspective. Acta Bot. Neerl., 45: 17-28.Direct Link |
Visser, E.J.W. and L.A.C.J. Voesenek, 2004.
Acclimation to soil flooding-sensing and signal-transduction: A review. Plant Soil, 254: 197-214.CrossRef | Direct Link |
Zaidi, P., S. Rafique and N. Singh, 2003.
Response of Zea mays
genotypes to excess soil moisture stress: Morpho-Physiological effects and basis of tolerance. Eur. J. Agron., 19: 383-399.Direct Link |
Zhang, J. and M.B. Kirkham, 1996.
Antioxidation responses to drought in Sunflower and Sorghum seedling. New Phytol., 132: 361-373.CrossRef |