Abstract: 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.
INTRODUCTION
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 |
| Chla | = Chlorophyll a |
| Chlb | = Chlorophyll b |
| A | = 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
| Fig. 1: | 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) |
| Fig. 2: | 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) |
| Fig. 3: | The roots of flooded Zea mays plants tend to become negatively gravitropic and adventitious roots develop against flooding |
| Fig. 4: | 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) |
| Fig. 5: | 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).
CONCLUSION
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.