Plants that are adapted to waterlogged conditions develop aerenchyma in roots for ventilation. Some wetland plant species also form an apoplastic barrier at the outer cell layers of roots that reduces radial oxygen loss (ROL) from the aerenchyma and prevents toxic compounds from entering the root. The composition of the apoplastic barrier is not well understood. One potential component is suberin, which accumulates at the hypodermal/exodermal cell layers of the roots under waterlogged soil conditions or in response to other environmental stimuli. However, differences in suberin content and composition between plant species make it difficult to evaluate whether suberin has a role in preventing ROL. In this article, we summarize recent advances in understanding apoplastic barrier formation in roots and, between various plant species, compare the chemical compositions of the apoplastic barriers in relation to their permeability to oxygen. Moreover, the relationship between suberin accumulation and the barrier to ROL is discussed.

Rice coleoptiles, renowned for anoxia tolerance, were hypoxically pretreated, excised, ‘healed’, and then exposed to a combination of anoxia and pH 3.5. The putative acid load was confirmed by net effluxes of K+ to the medium, with concurrent net decreases of H+ in the medium, presumably mainly due to H+ influx. Yet the coleoptiles survived the combination of anoxia and pH 3.5 for at least 90 h, and even for at least 40 h when the energy crisis, inherent to anoxia, had been aggravated by supplying the coleoptiles with 2.5 mM rather than 50 mM glucose. Even in the case of coleoptiles with 2.5 mM glucose, an accumulation ratio of 6 for Cl– was attained at 4 h after the start of re-aeration, implying plasma membrane integrity was either maintained during anoxia, or rapidly restored after a return to aerated conditions. Cytoplasmic pH and vacuolar pH were measured using in vivo 31P nuclear magnetic resonance spectroscopy with 50 mM glucose in the basal perfusion medium. After 60 h in anoxia, external pH was suddenly decreased from 6.5 to 3.5, but cytoplasmic pH only decreased from 7.35 to 7.2 during the first 2 h and then remained steady for the next 16 h. During the first 3 h at pH 3.5, vacuolar pH decreased from 5.7 to 5.25 and then stabilized. After 18 h at pH 3.5, the initial values of cytoplasmic pH and vacuolar pH were rapidly restored, both upon a return to pH 6.5 while maintaining anoxia and after subsequent return to aerated solution. Summing up, rice coleoptiles exposed to a combination of anoxia and pH 3.5 retained pH regulation and cellular compartmentation, demonstrating tolerance to anoxia even during the acid load imposed by exposure to pH 3.5.

The formation of a barrier to radial oxygen (O2 ) loss (ROL) in the root is an important adaptation of plants to root flooding, but the biochemical changes in plant roots where the barrier is formed are unclear. In this study, we analysed metabolic profiles and gene expression profiles in roots of rice (Oryza sativa L.) plants grown under stagnant deoxygenated conditions, which induce suberization in the outer cell layers of the roots and formation of barrier to ROL. Under these conditions, two distinctive biochemical features of the roots were the accumulations of malic acid and very long chain fatty acids (VLCFAs). We also showed that the expressions of some genes encoding plastid-localized enzymes, which convert malic acid to acetyl coenzyme A (AcCoA), were simultaneously up-regulated under stagnant conditions. The expression levels of these genes in specific root tissues isolated by laser microdissection suggested that malic acid is converted to AcCoA predominantly in the plastids in the outer cell layers of rice roots. We propose that the physiological role of malic acid accumulation in rice roots grown under stagnant conditions is to provide a substrate for the biosynthesis of fatty acids, which, in turn, are used in the biosynthesis of suberin.

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H+ influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K+ efflux was continuous. Further experiments used excised coleoptile tips (7–10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO3−, which distinguished two processes involved in pH regulation. Net H+ influx (μmol g−1 fresh weight h−1) for coleoptiles with NO3− was ∼1.55 over the first 24 h, being about twice that in the absence of NO3−, but then decreased to 0.5–0.9 as net NO3− uptake declined from ∼1.3 to 0.5, indicating reduced uptake via H+–NO3− symports. NO3− reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K+ balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO3− supply. Thus, biochemical pHstats and reduced net H+ influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.

No abstract

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH cyt and pH vac ) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH cyt and pH vac , glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice (Oryza sativa L.) root tips in comparison with those of wheat (Triticum aestivum L.) with in vivo 31 P-nuclear magnetic resonance. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6-7.7 to 7.1), which was followed by slow partial recovery (0.3 units after 6 h). Anoxia led to a dramatic pH cyt drop in tissues of both species (from pH 7.6-7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. In both plants, NTP content followed the dynamics of pH cyt . There was a strong correlation between NTP content and cytoplasmic H 1 activity ([H 1 ] cyt ¼ 10 2pH cyt ) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.

No abstract

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with (31)P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6-7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH(cyt) drop in tissues of both species (from pH 7.6-7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH(cyt). There was a strong correlation between NTP content and cytoplasmic H(+) activity ([H(+)](cyt) = 10(-pHcyt)) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader's attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.