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Plants are aerobic organisms and a lack of oxygen presents a severe stress. Low oxygen conditions in plants can be brought about by high metabolic activity, soil waterlogging or flooding. Soil waterlogging and flooding are environmental conditions that are frequently encountered by plants growing in aquatic or semi-aquatic habitats. Plant roots are most often exposed to a lack of oxygen as gas exchange is strongly decreased in waterlogged soils. However, when plant shoots become flooded, they also experience oxygen shortage. The problem of oxygen supply becomes more severe in turbid waters or with deep floods, which cause low light conditions that prevent photosynthetic oxygen production. Plants, such as rice (Oryza sativa), endure frequent flooding by adaptations found not only in roots, but also in stems. One basic strategy to cope with flooding is to improve gas exchange. Gas-filled spaces (aerenchyma) are constitutively found in aquatic and semi-aquatic plants, and are considered to be an efficient mechanism to ameliorate low oxygen stress. Aerenchyma facilitate gas exchange between aerial and submerged plant parts by reducing the diffusion resistance to gas exchange imposed by cells.
Aerenchyma can form by lysigeny or schizogeny. Lysigenous aerenchyma formation is observed in rice or maize roots, where cortex cells undergo programmed cell death. Subsequently, the cell corpses, including the cell walls, are degraded and resorbed, resulting in a gas space (Kawai et al., 1998; Evans, 2003). Aerenchyma form along the length of a plant organ, such as the root. In rice, they are also present in stems and leaves (Arashi & Nitta, 1955; Matsukura et al., 2000; Colmer & Pedersen, 2008). In wetland species, such as Sagittaria lancifolia and rice, which are well adapted to frequent flooding, aerenchyma develop constitutively (Raskin & Kende, 1983; Webb & Armstrong, 1983; Schussler & Longstreth, 1996). However, there is large variation in the development of adaptive traits, even among different rice cultivars (Fukao et al., 2006; Hattori et al., 2009).
Ethylene has been identified as the hormonal signal that mediates aerenchyma formation in wheat, maize, rice and Arabidopsis (Drew et al., 1981; Konings, 1982; Jackson et al., 1985; Justin & Armstrong, 1991; He et al., 1996; Watkin et al., 1998; Gunawardena et al., 2001; Mühlenbock et al., 2007; Shiono et al., 2008), whereas, in Juncus effuses, root aerenchyma form independent of ethylene (Visser & Bögemann, 2006). The diffusion rate of the gaseous hormone ethylene is greatly reduced on submergence, resulting in rapid accumulation in submerged tissues. Although the ethylene signaling pathway has been elucidated in detail, molecular events that result in the induction and execution of programmed cell death during aerenchyma formation are less well understood. In the maize root, Ca2+, protein phosphorylation and G-protein signaling appear to be integral signal components, as reagents that increase cytosolic Ca2+, protein phosphatase inhibitors and constitutive activation of G-protein promote aerenchyma formation at normoxic conditions (He et al., 1996). In rice roots, cell death has been shown to begin with the largest cell in the mid-cortex and to expand radially to neighboring cells (Kawai et al., 1998).
Programmed cell death is a developmental program that is indispensable for plant development, and represents an important strategy of plants for adaptation to biotic and abiotic stresses. The hypersensitive response (HR) describes local cell death in response to pathogen attack and helps to limit the spread of the pathogen (Zurbriggen et al., 2010). Epidermal cell death precedes the emergence of adventitious roots at the nodes of submerged rice plants. As is the case with aerenchymal cell death, epidermal cell death is induced by ethylene (Mergemann & Sauter, 2000). Hydrogen peroxide (H2O2) has been shown recently to act as a signal that is required for ethylene-induced epidermal cell death and is sufficient to promote it (Steffens & Sauter, 2005, 2009). Reactive oxygen species (ROS) have also been recognized as important signaling molecules in the hypersensitive cell death response (Lamb & Dixon, 1997). HR is characterized by an oxidative burst during which high levels of H2O2 are produced.
In this study, we analyzed aerenchyma formation in the rice stem. Based on the hypothesis that parenchymal cell death may be accompanied by ROS production, we analyzed the presence of H2O2 and the superoxide anion radical (O2·−) in the rice stem. Furthermore, we pursued the question of whether an increase in H2O2 would be sufficient to induce parenchymal cell death and thus promote aerenchyma formation. In addition to H2O2 treatment, we employed a rice mutant with an insertional mutation in the MT2b gene, which encodes a metallothionein. In rice suspension cells, constitutive genetic downregulation of MT2b transcript levels resulted in the accumulation of H2O2, showing that MT2b acts as a ROS scavenger (Wong et al., 2004). In addition, both hypersensitive cell death and epidermal cell death were enhanced in the MT2b::Tos17 mutant (Wong et al., 2004; Yuan et al., 2008; Steffens & Sauter, 2009). The data provided in this study show that aerenchyma formation is also controlled by H2O2, indicating that ROS play a key role in the regulation of diverse cell death processes in rice.
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Improved gas exchange is considered to be a hallmark of flooding tolerance, and is observed in essentially all wetland species. Improved gas exchange is achieved externally through leaf gas films that transport oxygen from leaves which remain above the water surface to submerged plant parts (Raskin & Kende, 1985), or that enlarge the gas–water interface in completely submerged plants (Pedersen et al., 2009). Improved gas exchange is further achieved through internal air spaces. As soil waterlogging or flooding affects primarily the roots, much research has focused on the study of aerenchyma formation in roots (e.g. Raskin & Kende, 1983; Webb & Armstrong, 1983; Schussler & Longstreth, 1996; Colmer, 2003). However, aerenchyma formation also occurs in stems and other shoot organs, where it may be especially important during deep floods when shoots become partially submerged, such as in rice (Arashi & Nitta, 1955). In this study, developmental and submergence-induced aerenchyma formation was analyzed in the rice stem. The deep-water rice variety PG56 showed a higher percentage of aerenchyma formed constitutively than the lowland rice varieties Kinmaze and Nipponbare, with the total number of aerenchymal tracts being the same in the two varieties. In the rice stem, aerenchyma were formed in between the vascular bundles which were located in an outer ring below the epidermis. The conserved number of aerenchyma may be a result of the conserved anatomy of rice with a defined number of vascular bundles in the stem.
The number of aerenchyma formed increased from bottom to top in a recurrent pattern that was observed in each internode. Nodes did not display gas spaces. Aerenchyma are constitutively formed in many aquatic or semi-aquatic plant species. On flooding, enhanced aerenchyma formation is observed in (semi-)aquatic, but also in nonaquatic species, as shown for maize (Drew et al., 1989; He et al., 1994), Spartina patens (Burdick, 1989), Luffa cylindrica (Shimamura et al., 2007) and Rumex palustris (Laan et al., 1991; Pierik et al., 2009), to name only a few. However, not all plants can form aerenchyma, as reported for Brassica napus (Voesenek et al., 1999). Consequently, the porosity value in aerated rapeseed roots is 3%, whereas rice roots show a constitutive porosity of 25–30% (Armstrong, 1971). During submergence, the porosity value remains constant in rapeseed and increases to 40–50% in rice roots. In the rice stem, partial submergence for 3 d resulted in an increase in aerenchyma formation throughout all internodes, indicating that all pre-aerenchymal cells were responsive to the cell death and cell lysis promoting signal. Despite the overall increase in aerenchyma numbers following submergence, a gradient remained in each internode, with fewer aerenchyma at the base than at the top. For instance, twice as many aerenchyma were counted at distances of 30 and 40 mm than at 10 mm above the third node. This gradient within the stem is exacerbated by the fact that nodes do not form any aerenchyma, indicating that neither aerenchyma nor the central pith cavity form a continuous gas space even when submerged. It is, however, conceivable that gas flow across the node occurs through intercellular spaces. Gas flow measurements, such as were performed in Phragmites australis (Afreen et al., 2007), will be required to determine the actual resistance to gas diffusion in the internodes and at the stem nodes of rice. It has been proposed that aerenchyma not only improve gas exchange, but also reduce the number of cells requiring oxygen for respiration (Kawai et al., 1998). In maize roots, aerenchyma form even under drought conditions (Zhu et al., 2010). Recombinant inbred maize lines with high root cortical aerenchyma produced higher shoot biomass and more seed when grown under water-stressed (drought) conditions than did lines which formed very few aerenchyma. The improved drought tolerance in lines with high aerenchyma formation has been proposed to be a result of reduced metabolic cost in the roots caused by reduced cell numbers (Zhu et al., 2010).
A more detailed analysis of the cells that are lysed to form a gas space in the rice stem revealed that these cells possess characteristic features. They are larger, as also observed for cortex cells in rice roots that form aerenchyma (Kawai et al., 1998). They contain a large vacuole, are lighter in color and appear to be devoid of chloroplasts. Unlike the surrounding parenchymal cells, pre-aerenchymal cells contain little or no starch, and have a thin cell wall. By contrast, the walls in cells that surround aerenchyma are thicker, an observation that was also made in other (semi-)aquatic species, such as Astrocaryum jauari (Schlüter et al., 1993). Thick cell walls around an aerenchyma are thought to prevent its collapse, as the hydrostatic pressure increases with water depth. In rice internodes, pre-aerenchymal cells were occasionally stained with Evans blue, indicating that they had undergone cell death, but were not yet lysed. Overall, cells that eventually form gas spaces in the rice stem do not appear to participate greatly in photosynthetic activity, or contribute to tissue stability, raising the question of why they are maintained to begin with.
Aerenchyma formation in response to submergence has been shown previously to be regulated by ethylene in wheat (Watkin et al., 1998) and maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985; He et al., 1996; Gunawardena et al., 2001) roots. In rice, ethylene can promote aerenchyma formation in adventitious roots (Justin & Armstrong, 1991), and in internodes, as shown in this study. To date, little else is known about cell death regulation and lysis of cells during gas space formation. ROS have been recognized as central components of cell death signaling (Kotchoni & Gachomo, 2006). Cadmium-induced death of suspension cells of tomato was enhanced by ethylene and was accompanied by elevated levels of H2O2 (Yakimova et al., 2006). In rice, death of epidermal cells above nodal adventitious root primordia was induced by ethylene and H2O2 (Steffens & Sauter, 2009). Pre-aerenchymal cells in the rice internode were characterized by elevated levels of superoxide anion radical and H2O2 when compared with surrounding cells. Induction of aerenchyma formation by ethylene was preceded by an increase in O2·−, reminiscent of an oxidative burst, at c. 16 h that preceded ethylene-induced aerenchyma formation. In the mid-cortex of maize roots, aerenchyma formation induced by sulfate starvation was similarly preceded by O2·− and H2O2 generation that was observed in some of these cells (Bouranis et al., 2003). Epidermal cell death in rice was also promoted when endogenous levels of H2O2 were increased by the inhibition of catalase activity. Similarly, exogenous application of H2O2 promoted epidermal cell death, even in the presence of 1-methylcyclopropene, which inhibits ethylene perception, indicating that H2O2 acts downstream of ethylene.
Steady-state levels of ROS result from a balance between synthesis and scavenging. ROS detoxification is achieved enzymatically, for example by ascorbate peroxidase, superoxide dismutase or catalase, or nonenzymatically (Jwa et al., 2006). In rice, the metallothionein MT2b acts as a scavenger of H2O2 (Wong et al., 2004). Genetic downregulation of MT2b in rice promoted epidermal cell death and hypersensitive cell death in leaves (Wong et al., 2004; Steffens & Sauter, 2009), and enhanced submergence-induced aerenchyma formation in the stem. These findings support the view that altered ROS scavenging is a highly efficient means to alter ROS levels, such that a physiological cell death response follows. In epidermal cells above adventitious root primordia, MT2b was shown to be downregulated in response to ethylene or H2O2, indicating that reduced ROS scavenging through altered MT2b activity may be a general mechanism of cell death regulation in rice.