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- Materials and Methods
When faced with oxygen depletion in flooded soil, rice (Oryza sativa L.), like other wetland species, develops numerous morphological and biochemical responses to reduce the impact of the stress. The aerenchyma is of particular importance. It facilitates the internal transport of gasses from well-aerated aerial shoots to underground organs exposed to anaerobic surroundings. The effectiveness of the aerenchyma can be increased by the formation of barriers in the outer cell layers of roots. These barriers inhibit radial O2 loss (ROL) to the rhizosphere and enhance longitudinal diffusion of O2 towards the root apex (Armstrong, 1979; Colmer, 2003b).
Roots of some wetland species constitutively express barriers to ROL (e.g. Juncus effusus and Eleocharis acuta; Visser et al., 2000; McDonald et al., 2002). In others, barriers are induced during growth in stagnant, deoxygenated media (e.g. Oryza sativa, wild Hordeum species and Lolium multiflorum; Colmer et al., 1998; McDonald et al., 2002; Colmer, 2003a; Garthwaite et al., 2003; Kotula et al., 2009). The barrier to ROL in roots has usually been linked to suberization and lignification of the walls of the peripheral cell layers (De Simone et al., 2003; Soukup et al., 2007; Garthwaite et al., 2008). Kotula et al. (2009) combined measurements of ROL with histochemical and biochemical analyses of the outer part of roots (OPR) of rice. They showed that, when grown in deoxygenated solution, the amounts of suberin and lignin in OPR sleeves were several folds greater than those of plants grown in aerated solution. This correlated with the pattern of ROLs. It was concluded that the suberized exodermis and lignified sclerenchyma of rice roots formed a strong barrier to ROL.
Despite the importance of the barrier to ROL for roots of wetland plants, there are few data available on the permeability coefficient of O2 across cell layers exterior to the aerenchyma. In Phragmites australis, this permeability coefficient was assessed using O2 concentration profiles across the hypodermis/epidermis, in combination with rates of O2 consumption in these layers (Armstrong et al., 2000). Using roots of Hordeum marinum, Garthwaite et al. (2008) developed another method to determine the diffusivity of O2 across outer cell layers. Diffusivities were derived from measurements of ROL obtained while either varying shoot O2 partial pressure or cooling the rooting medium to cancel respiratory effects. In the present study, the O2 permeability coefficient of the OPR (POPR) of rice has been measured directly using the method recently developed by Kotula & Steudle (2009). The technique was based on perfusing the aerenchyma of root segments with gas mixtures (O2:N2) of known O2 concentration while, at the same time, measuring radial losses of oxygen using a root-sleeving platinum electrode (Armstrong, 1994). The O2 permeability of the OPR could be calculated, as both the O2 flow across the OPR and the O2 concentration gradient between the aerenchyma and the surrounding medium were known.
In contrast to the previous study of Kotula & Steudle (2009), where plants were grown in an aerated solution, in the present study we examined the effects of different growth conditions on POPR by growing rice in either aerated or stagnant, deoxygenated medium. In order to estimate the contribution of the apoplast and living cells to the overall movement of O2 across the OPR, the POPR was varied either by partially blocking apoplastic pores in the OPR with salt precipitates or by killing living tissue with 0.1 N HCl. As far as we are aware, this is the first direct quantitative comparison of O2 permeability coefficients of peripheral layers in roots subjected to aerated or deoxygenated treatments. Our study provides evidence of the effectiveness of apoplastic barriers in reducing O2 and ion permeability across the root outer cell layers, when plants are grown in deoxygenated medium.
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- Materials and Methods
In the present study, the gas perfusion technique developed by Kotula & Steudle (2009) was used to measure O2 permeability coefficients of the outer cell layers of roots (POPR) of rice plants grown in either aerated or stagnant, deoxygenated conditions. As far as we are aware, this is the first quantitative comparison of the permeabilities of the peripheral layers to O2 in rice roots and how these change when the OPR becomes modified. The results indicated that, when plants were grown in the stagnant deoxygenated solution, the POPR of all investigated zones was several folds smaller than that of plants grown in the aerated solution. When salt precipitates were formed in cell wall pores in roots of plants grown under aerated conditions, O2 permeability decreased. By contrast, POPR increased in response to the killing of root cells with 0.1 N HCl. The effect of the latter treatment was relatively small in roots in the aerated solution but larger in roots in the stagnant medium, suggesting a significant contribution of respiratory effects in the presence of low POPR. Overall, POPR was strongly affected by the apoplastic barriers in the roots of rice, which lowered oxygen diffusion across the peripheral cell layers.
The lower POPR in plants grown in the stagnant medium as well as the reduction of POPR along the roots of plants grown in both conditions strongly correlated with the development of apoplastic barriers in the OPR. In a quantitative examination, Kotula et al. (2009) showed that the amounts of suberin and lignin increased along the roots of rice plants grown in both aerated and stagnant solutions; however, absolute loads of these polymers were much greater in roots grown under deoxygenated conditions. In agreement with chemical analyses, detailed histochemical studies of the OPR of rice revealed early development of exodermal Casparian bands and suberin lamellae in plants grown in stagnant conditions. In addition to suberization, early lignification of walls of densely packed sclerenchyma was found closer to the root apex in these plants (Kotula et al., 2009). Apparently, apoplastic barriers impeded gas diffusion across the OPR. Suberin, in particular, is known to offer a high resistance to O2 diffusion (De Simone et al., 2003; Soukup et al., 2007). The present study supports these views.
Compared with the diffusion of water, O2 diffusion across the OPR of plants grown in the aerated solution was greater by an order of magnitude. This may result from the fact that, in contrast to the O2 molecule, water has a polar (dipole) structure, tending to reduce its diffusivity in suberized cell walls. Similar differences were found for cuticles (Lendzian, 1982; Lendzian & Kerstiens, 1991). The bulk (hydraulic) water permeability across the OPR of rice grown in an aerated solution was much greater than diffusional water flow (Ranathunge et al., 2004). Modification of the apoplast of the OPR by filling intermicrofibrillar spaces (cell wall pores) with precipitates reduced the diffusional permeability of both water and O2 by c. 20%. By contrast, this treatment caused a massive three- to four-fold reduction of hydraulic conductivity (LpOPR; Ranathunge et al., 2005). This indicated that precipitates affected bulk flows of water much more than diffusive flows of water and oxygen. In contrast to bulk water flow, the diffusion of O2 across outer cell layers ought to be appreciable over the whole inter-cell interface and was not restricted by cell membranes, which are thought to be highly permeable to O2 (Armstrong, 1979; Nobel, 2005). The diffusion of O2 across the OPR should be limited by the existence of apoplastic barriers such as suberin lamellae and Casparian bands. Hence, the OPR allows rather high water flow in the presence of a relatively high resistance to O2. This is achieved through differences in transport mechanisms: there is a bulk flow of water, but the flow of O2 is diffusional in nature (Kotula & Steudle, 2009). This suggested that rice has evolved an optimum balance between water uptake and O2 loss, with high rates of water uptake in the presence of low rates of O2 loss (Ranathunge et al., 2004; Kotula & Steudle, 2009). However, to date, the above conclusion had been reached only for rice grown in aerated conditions. When plants were grown in a stagnant deoxygenated solution, which mimics natural paddy-field conditions, roots showed several-fold greater amounts of suberin and lignin in the OPR and drastically reduced POPR. This means that pores were either completely absent in the apoplastic barriers under these conditions or the diameters of pores were rather small. This situation differs from that in the cuticles, for which the precipitation technique of Ranathunge et al. (2005) has been used to demonstrate the existence of pores (Schreiber et al., 2006). In future studies, the existence or absence of pores in apoplastic barriers of roots of plants grown under deoxygenated conditions needs to be determined by comparing the diffusional and bulk permeabilities of water.
In agreement with Ranathunge et al. (2005), precipitation treatment of roots from aerated hydroponics produced intense brown precipitates at the apical parts of the root segments which gradually faded along the root towards the base. At least in the immature parts of roots, ions could pass through the exodermis and sclerenchyma layer. Because of the development of apoplastic barriers, ion movement across the OPR was reduced in the more mature parts, but was still present. At distances of 50–60 mm, salt precipitates revealed a patchy structure, which may correlate with the maturation of the exodermis (Fig.2d). In cross-sections of salt-treated roots, brown precipitates were localized to the side where K4[Fe(CN)6] was applied, suggesting that Cu2+ rather than [Fe(CN)6]4− passed through the barrier of the OPR in plants grown in aerated conditions. Obviously, the ferrocyanide anion, with its four negative charges, moves across the barrier much more slowly than the positively charged copper cations, probably because of repulsion by the negative fixed charges of the cell wall matrix. In contrast to plants grown in aerated conditions, no precipitates were observed in the roots of plants grown in the deoxygenated medium. This is evidence that well-developed apoplastic barriers in the exodermis and lignified fibre cells impeded ion movement across the OPR. The concentration of Cu2+ and [Fe(CN)6]4− ions in the apoplast remained too low to cause precipitation. In addition, suberin lamellae may block ion transport through membranes. It was already suspected that the suberization/lignification of rice roots may form a barrier that reduces the uptake of ions, such as Fe2+ (Armstrong & Armstrong, 2005). Colmer & Bloom (1998) showed that NH4+ and NO3− uptake in basal regions of roots of O. sativa was c. 30% of that in Zea mays, even when plants were grown in aerated solution. The present results suggest that the barrier to ROL, which is induced during growth under deoxygenated conditions, reduces the transport of ions. Under stagnant conditions, the number and size of pores in the apoplast may also be reduced.
When cell wall pores were blocked with precipitates, the greatest reduction in POPR was observed close to the root apex, where the exodermis was not yet fully developed. The effect was reduced at the more basal parts of the root. The dense precipitates formed in the OPR hindered radial O2 diffusion through the transcellular pathway, which is also done by suberin lamellae in well-developed roots (Ranathunge et al., 2005). However, it should be noted that the precipitates may not have completely blocked the pores in the cell walls. Thus, the reduction in O2 permeability was relatively small. Overall, the effect of salt precipitates was twofold: firstly an additional barrier was created in series to the apoplastic barriers, and secondly pores within the barriers may have been blocked. No reduction in O2 permeability in plants grown in the deoxygenated solution resulted from the lack of salt precipitates in cell wall pores.
At all tested distances and growth conditions, the acid treatment caused a relatively small increase in POPR. The increase may have resulted from cancelling of respiratory O2 consumption by living cells in the OPR. It was possible to estimate the respiration rates of the OPR from the differences in radial O2 flows (in nmol m−2 s−1) between control and HCl-treated roots. Assuming the thickness of the OPR to be 85 μm and the root diameter to be 1 mm, the respiration rate of a 40-mm-long root segment at atmospheric O2 concentration would be 3.9 × 10−11 and 2.6 × 10−11 g O2 mm−3 s−1 for plants grown in aerated and deoxygenated solutions, respectively (for a ref. see Kotula and Steudle, 2009). Although there was a higher respiration rate in plants grown in the aerated solution, the relative increase in POPR after the cells had been killed was smaller than that found in plants grown in the stagnant medium. This suggested that, in plants grown in the aerated solution, the effect of respiration on POPRshould be relatively small in relation to high rates of JO2. This may differ from the situation in plants grown in stagnant conditions, where JO2 is relatively low. The data indicated that respiratory O2 consumption contributed much more in conjunction with a reduced O2 permeability. It may be argued that, in addition to the respiratory activity, the acid treatment may affect the integrity of the apoplastic barrier by causing disintegration of the suberin or lignin polymers, allowing higher O2 diffusion. However, as both suberin and lignin are known to be chemically resistant (Johansen, 1940; Ranathunge et al., 2008), 0.1N HCl probably only had a small effect on them. This conclusion agrees with the findings that, firstly, the POPR of HCl-killed roots grown in the deoxygenated solution was still significantly smaller than that of untreated roots grown in the aerated solution and, secondly, in root segments from plants grown in stagnant conditions, no precipitates were observed after killing with HCl. Overall, the ‘physical resistance’ played a dominant role in impeding O2 loss from rice roots, although respiration may also have an effect, when rates of radial O2 loss were low. The results support the findings of previous studies by Armstrong et al. (2000) and Garthwaite et al. (2008) on roots of Phragmites australis and Hordeum marinum, respectively. In H. marinum, the physical barrier appeared to account for 84% of the reduction and respiratory activity for 16% (Garthwaite et al., 2008).
In conclusion, the quantitative comparison of O2 permeability coefficients across the OPR (POPR) of rice grown in either an aerated or a deoxygenated solution indicated that the formation of apoplastic barriers strongly decreased POPR. Treatment with CuSO4/K4[Fe(CN)6] resulted in the formation of brown precipitates only in the roots of plants grown in the aerated solution, indicating that the apoplast of the OPR has a somewhat porous structure. By contrast, no precipitates were observed in roots of plants grown in the deoxygenated medium. It was concluded that well-developed apoplastic barriers in the exodermis, such as Casparian bands and suberin lamellae, as well as lignified fibre cells, blocked ion movement across the OPR. Even after killing of the root segments of plants grown in stagnant conditions, no brown precipitates were observed. This confirmed that apoplastic barriers had a substantial effect on ion movement. The formation of precipitates in the OPR of aerated plants reduced POPR by 5 to 20%, depending on the development of the apoplastic barriers along the root. Comparison with earlier findings concerning diffusional and hydraulic water permeabilities supports the view that apoplastic barriers provided substantial resistance to the diffusion of O2 across the OPR but allowed high bulk water flow. It appears that, in rice, the OPR allows rather high water flow in the presence of a high resistance to O2, which is an advantage for the plant. Killing of root cells by dilute hydrochloric acid increased POPR by up to 55%. This suggested a significant, although not dominant role of respiratory O2 consumption in living cells of the OPR. The fact that the POPR of HCl-treated roots of plants grown in the deoxygenated solution was still significantly lower than that of untreated aerated plants further supports the view that suberization and/or lignification provides a strong barrier to O2 flow across the OPR of rice.