Plants deploy a number of strategies to survive under variable environmental conditions. Stresses such as drought and salinity may have devastating effects on plant survival. The shoot is the first part of the plant to feel drought stress, as it is the location of water loss. Salinity, on the other hand, would affect the roots first, since they are in direct contact with the soil. Consequently, it may be expected that roots would display adaptations to cope with variation in soil salinity. The hydraulic conductivity of roots of both herbaceous and woody species has been extensively investigated (Steudle and Heydt, 1997
; Barrowclough et al., 2000
; Miyamoto et al., 2001
; Lee et al., 2004
). These studies indicate that unfavourable environmental conditions reduce hydraulic conductivity (Steudle, 1994
; Kramer and Boyer, 1995
; Steudle and Peterson, 1998
; Zimmermann et al., 2000
). Suberization of roots such as formation of CBs and suberin lamellae localized in cell walls, as those of the endo- and exodermis contribute to the observed variability of water uptake in roots, with more extensive barriers effectively reducing conductivity (Cruz et al., 1992
; Stasovsky and Perterson, 1993
). However, all barrier structures do not contribute equally to the reduction in hydraulic conductivity. The exodermal barrier, for instance, has been shown to present relatively little resistance to water flow in maize and rice (Zimmermann and Steudle, 1998
; Ranathunge et al., 2003
). In rice, it is the highly suberized endodermal barrier that presents the major resistance to radial water flow (Miyamoto et al., 2001
; Ranathunge et al., 2003
). Further, the chemical composition of suberin in the apoplastic barriers is reported to affect the hydraulic conductivity of roots (Schreiber et al., 2005
One of the hydrophobic barriers, namely CBs, serve to prevent transport of the external medium including ions and fluorescent dyes directly into the xylem stream, effectively ensuring that fluid has to pass through at least one cell membrane en route (Steudle and Peterson, 1998
). This strategy, in turn, allows for some control of solutes and fluid that are transported to the shoot via the xylem stream. Most of the Na+
that enters the shoots of rice plants has been reported to do so through the so-called ‘apoplastic bypass’, where Na+
ions move through the apoplast by solvent drag (Ranathunge et al., 2005
), bypassing CBs (Ochiai and Matoh, 2002
; Gong et al., 2006
). Exposure of rice to moderate saline stress of 100 mM NaCl for 1 week resulted in deposition of additional barrier material (i.e. suberin), strengthening the apoplastic barriers (Krishnamurthy et al., 2009
). The present study was designed to test whether or not these induced barriers resist the bypass flow, thereby contributing to a reduction in Na+
uptake and hence enhance survival of rice plants that remain in a saline environment.
In the absence of stress, the sensitive cultivar, IR20, exhibited significantly higher hydraulic conductivity than did the tolerant Pokkali ()—in good agreement with earlier results on the suberin contents of the respective roots (Krishnamurthy et al., 2009
). Subjecting the plants to a moderate conditioning stress of 100 mM NaCl resulted in a large reduction in hydraulic conductivity, which is also consistent with data on the deposition of additional suberin during this period and formation of the CBs in the endo- and exodermis close to the root tips (). The extent of barrier deposition (i.e. the fold increase in total root suberin) was significantly greater for IR20 than for Pokkali (Krishnamurthy et al., 2009
). Indeed the reduction of hydraulic conductivity was also more dramatic for IR20 than for Pokkali ().
The volume flow (Jvr
) of water through the root increased with applied pneumatic pressure in a non-linear fashion (). In the absence of applied pneumatic pressure, Jvr
is exclusively due to osmotically driven movement of xylem sap. As the applied pressure is increased, an increasing fraction of the fluid collected in the experiment is due to hydrostatic pressure-driven flow through the apoplastic route (i.e. xylem sap is diluted by the Fiscus Effect; Fiscus, 1975
; Miyamoto et al., 2001
). At high hydrostatic pressures, Jvr
is dominated by apoplastic water movement and the relationship is linear. The hydraulic conductivity, Lpr
, was estimated from this linear part of the curve and is thus expected to reflect apoplastic water movement. Osmotically driven water flow moves primarily through plasma membranes and requires the activity of aquaporins. Gating of aquaporins is known to be osmotically regulated (Ye et al., 2004
; Lee et al., 2005
). Hence, salt-stressed plants were transferred to control medium for 12 h prior to starting water permeability measurements to avoid osmotic stress-induced closure of water channels. On the other hand, deposition of suberin lamellae very close to the root tips of these cultivars was observed following a conditioning salt stress of 100 mM in an earlier study (Krishnamurthy et al., 2009
). These barriers could have contributed to the inability to observe osmotically driven fluid flow in the stressed plants.
The performance of the barriers laid down during conditioning differed from the pre-existing barriers in terms of the pore size distribution. Bypass of Na+ and PTS was similar in unstressed roots of both cultivars, indicating that the cell wall pores were too large to distinguish between Na+ and PTS. On conditioning, bypass reduced significantly for these solutes in both cultivars (, B). This reduction in bypass flow during conditioning suggests that either the total number of pores or their size is significantly reduced, probably both. Bypass of PTS was more severely curtailed than that of Na+, suggesting that the newly deposited suberin clogged the intermicrofibrillar spaces in the cell walls, making tight suberized barriers with reduced pore sizes. A significant fraction of the pores in the pre-existing barriers are much larger than the diameter of PTS, as the drag on PTS and Na+ is similar. However, the freshly deposited suberized barriers seem to sieve PTS more effectively than Na+, indicative of a pore size distribution with relatively few pores exceeding the diameter of PTS.
In rice, it has been suggested that K+
enter the shoot by distinct mechanisms which are genetically regulated (Garcia et al., 1997
). The present data show that the K+
content of the shoots is invariant across all the conditions imposed in this study, while the Na+
content in shoots varies widely (, C). The size of the pores bypassing the hydrophobic barriers is much larger than the diameter of the Na+
ion both before and after conditioning. The largest pores both before and after conditioning are larger than the diameter of PTS, and so cannot distinguish between Na+
. It follows that the amount of K+
taken up by the more specific and better regulated mechanism of loading endodermis and xylem via transporters in the plasma membrane greatly exceeds the amount entering through apoplastic bypass flow.
The present estimates of the hydraulic conductivity and bypass flow of unstressed (control) plants are somewhat higher than those previously reported for rice, but in the same range (Miyamoto et al., 2001
; Ranathunge et al., 2003
). Care was taken to handle the roots very gently and carefully to ensure that the high values obtained were not caused by artificial injuries or physical damage. In addition, similarly handled roots of stressed plants had significantly lower hydraulic conductivity, which fall well within the range reported. Further, it is known that rice plants exhibit enormous individual variability in bypass flow and Na+
uptake (Yeo and Flowers, 1983
). It is thus likely that the values reported here are higher than those in earlier studies because of varietal differences or growth conditions or the age of the preparations used. Varietal differences are clearly substantial as Lpr
for unstressed IR20 was almost double that seen for Pokkali (). Growth conditions are also critical inasmuch as Lpr
of IR20 dropped by a factor of ~2 after conditioning. On the other hand, not all cultivars are equally sensitive to salinity, as Pokkali Lpr
was reduced by only a third (33%) under the same conditions. Indeed, the final Lpr
for both cultivars after exposure to 100 mM NaCl was essentially indistinguishable.
Suberized, hydrophobic barriers present the major resistance to radial flow of water, and a good correlation was seen between the earlier report on the deposition of these barriers in IR20 and Pokkali (Krishnamurthy et al., 2009
) and formation of CBs () and the hydraulic conductivity measurements conducted in the present study. However, it has been suggested that the locations where lateral roots emerge from primary roots are leaky to water as barriers are interrupted at these points, presenting ‘open windows’ for water and solute flows (Peterson et al., 1981
; Ranathunge et al., 2004
). PTS leak into the cortex was seen at the primary root–lateral root junctions where the lateral roots emerge. On the other hand, no such leak was found at the base of mature lateral roots, where the wound created due to lateral root emergence could have healed (). This finding disagrees with that of Faiyue et al. (2010a)
who have reported PTS entry into the xylem. However, they also suggested that the emerging lateral roots themselves may admit Na+
as they lack an exodermis (Faiyue et al., 2010b
). On the other hand, the exodermis plays a relatively small role in restricting water and solute entry into the root compared with the endodermis (Miyamoto et al., 2001
; Ranathunge et al., 2003
). Very prominent CBs were seen in the endodermis of lateral roots in control as well as stressed rice plants (), which could be involved in restricting the bypass flow of Na+
into the root xylem.
The present data indicate a significant increase in the density of lateral roots on exposure to salinity, together with a dramatic reduction in hydraulic conductivity and bypass flow. This would suggest that the contribution of lateral roots, if any, to bypass flow is small compared with the resistance presented by the newly deposited hydrophobic barriers in the primary root. Functionally, the extent of Na+ uptake correlates very well with the hydrostatic hydraulic conductivity, suggesting that uptake of Na+ through exposed surfaces of lateral roots and subsequent cell to cell movement is negligible in this context.
While the emergence of lateral roots did not appear to increase the extent of bypass flow, the number of lateral roots emerging on exposure to salinity was significantly greater than in control plants. The total number of lateral roots initiated during stress was significantly higher than in control plants under all stress protocols used. Indeed, a significant enhancement in initiation is seen following the 2 d, acute stress protocol. However, emergence of these roots from the primary root appears to take >2 d inasmuch as the number of emerged lateral roots was much greater after a week of stress compared with a 2 d stress protocol (). This observation of increased lateral root density following stress is consistent with earlier reports on Arabidopsis
(Nibau et al., 2008
). It is conceivable that this increase is a means of combating the impairment of root hydraulics caused by the extensive suberization.
The deposition of suberized hydrophobic barriers during conditioning is well correlated with a sharp reduction in Na+ uptake into shoots during subsequent exposure to acute stress. Indeed, the present data indicate that the entry of Na+ into shoots under these conditions is reduced by almost an order of magnitude compared with the uptake on directly exposing plants to 200 mM NaCl (, B, III–I versus IV–II). Barrier function was still good 1 week after returning the plants to control medium (, VI–V versus III–I). It may be expected that this would be reflected in enhanced survival. Survival of plants conditioned with 100 mM NaCl stress was significantly better on subsequent exposure to toxic 200 mM NaCl compared with unconditioned plants (, C). In fact, there appears to be little additional mortality on exposure to acute stress after the conditioning ().
Interestingly, a significant fraction of the Na+
taken up into the shoot in the course of the conditioning protocol was released on return to control medium (, V; B, II–V). The amount of Na+
released was larger in the case of Pokkali than for IR20, but it was significant in both cases. The amount of Na+
released over a week of recovery was comparable with the amount of Na+
taken up into the shoots with an acute stress of 200 mM NaCl for 2 d after conditioning in both the cultivars (, II–V versus IV–II). Mechanisms for reduction of shoot Na+
may include efflux through the phloem back into the root, and exudation from the hydathodes of leaves. Subjecting plants which had gone through a recovery period of a week after the conditioning to an acute stress of 200 mM NaCl resulted in Na+
levels in the shoot comparable with those of plants subjected to acute stress without pre-conditioning (, VI and III). However, the survival of the conditioned plants was better than that of plants that had not been conditioned (). It was previously shown that survival is best correlated not with total shoot Na+
content, but with the Na+
content of the apoplast fraction of the shoot (Anil et al., 2005
). The apoplastic fraction of shoot Na+
for IR20 was lower in plants stressed after recovery from conditioning [14 mg g−1
fresh weight (FW)] than in plants subjected to 200 mM NaCl stress without prior conditioning (~18 mg g−1
FW) (). The correlation between the survival of plants of both cultivars subjected to the range of treatments studied here with their shoot apoplastic Na+
contents is presented in . Irrespective of stress protocol or cultivar, the negative correlation holds very well. Thus, the conditioning protocol served not only to build up hydrophobic barriers, but also to activate mechanisms for partitioning Na+
that enters the shoot in a manner that reduces its presence in the shoot apoplast.
Candidate genes for the regulation of lateral root growth include those for auxin transport, RAU1
. These correspond to the AUX1
-like genes in Arabidopsis
, which are involved in phloem-based indole acetic acid (IAA) transport (Marchant et al., 2002
; Chhun et al., 2007
). The present data indicate that neither of these genes is responsible for the initiation of lateral root development under stress in rice as RAU1
levels were invariant under the stress protocols used, while RAU4
levels declined under stress (). It is conceivable that other transporters play a role in auxin transport under these circumstances. However, the transcript levels of Arf8
were up-regulated upon salt stress, indicating a role for this in lateral root development of rice ().
In conclusion, the present data indicate a good correlation between apoplastic barrier deposition and resistance to radial flow of water and solutes in the roots of rice. Further, it is demonstrated that the apoplastic barriers deposited under moderate salinity stress (conditioning protocol) resist the flow of bulk water and dissolved solutes, resulting in reduced uptake of Na+ into shoots and consequently better survival under subsequent acute stress. These salinity-induced barriers have a pore size distribution with relatively few pores greatly exceeding the diameter of the apoplastic tracer dye, PTS, whereas the pre-existing barriers have much larger pores. Finally, while salinity stress induces the emergence of lateral roots, these do not appear to play a significant role in enhancing Na+ uptake into shoots.