Apoplastic pH during low-oxygen stress in Barley

Abstract

Background and aims: Anoxia leads to an energy crisis, tolerance of which varies from plant to plant. Although the apoplast represents an important storage and reaction space, and engages in the mediation of membrane transport, this extracellular compartment has not yet been granted a role during oxygen shortage. Here, an attempt is made to highlight the importance of the apoplast during oxygen stress and to test whether information about it is transferred systemically in Hordeum vulgare.

Methods: Non-invasive ion-selective microprobes were used which, after being inserted through open stomata, directly contact the apoplastic fluid and continuously measure the apoplastic pH and changes to it.

Key results: (a) Barley leaves respond to oxygen stress with apoplastic alkalinization and membrane depolarization. These responses are persistent under anoxia (N2; O2 < 3%) but transient under hypoxia. (b) Being applied to the root, the information 'anoxia' is signalled to the leaf as an increase in pH, whereas 'hypoxia' is not: flooding of the roots within the first 2 h has no effect on the leaf apoplastic pH, whereas anoxia (N2) or chemical anoxia (NaCN/salicylic hydroxamic acid) rapidly increase the leaf apoplastic pH. (c) Under anoxia, the proton motive force suffers a decrease by over 70 %, which impairs H(+) -driven transport.

Conclusions: Although anoxia-induced apoplastic alkalinization is a general response to stress, its impact on the proton motive force (reduction) and thus on transport mediation of energy-rich compounds is evident. It is concluded that anoxia tolerance depends on how the plant is able to hold the proton motive force and H(+) turnover at a level that guarantees sufficient energy is harvested to overcome the crisis.

Fig. 1

Principle of apoplastic pH measurements. (A) On a leaf of an intact plant. The roots were placed in an Erlenmeyer flask. The leaf was fixed with double-adhesive tape on a dry Plexiglass cuvette beneath the electrodes. The (cut) leaf tip rested in a chamber that held the exchangeable reference solution, but also harboured the earth electrode. Exchange of fluid (e.g. CN⁻) or gas flow (air, N₂) was achieved through pipes while recording. An O₂-minielectrode constantly controlled the O₂. (B) On a detached leaf. The cuvette consists of two chambers: a wet (reference) chamber, which holds an exchangeable solution and the earth electrode. Added agents (e.g. FC or cyanide) were transported to the site of measurement through the transpiration stream. The leaf blade was fixed and the electrodes were placed in the dry chamber, as described in (A). An extra gas-flow cuvette with a small opening at the top to allow the approach of the electrodes could be placed on top of the leaflet. As in (A), an oxygen electrode constantly measured the O_2.

Fig. 2

Apoplastic pH of barley leaves responding to 10 μm FC or to 5 mm NaCN + 0·2 mm salicylic hydroxamic acid (SHAM). Agents were added to the leaf according to Fig. 1B and reached the measuring site after being carried through the xylem stream, as described in Felle et al., (2000). The curves are representative of three experiments (FC) or four experiments (NaCN/SHAM).

Fig. 3

Apoplastic pH of barley leaves responding to infiltration. (A) Local infiltration through nanolitre pressure infusion with a solution that comprised 5 mm KCl, 0·1 mm CaCl₂. Curve 1: only the immediate area around the measuring site was infiltrated, as detected by microscopic observation. Curve 2: a larger area was infiltrated. (B) Voltage responses to a ‘light off’ test following leaf infiltration according to pattern 2, or without infiltration (control). Representative curves of three equivalent tests each. For experimental details, see Materials and Methods.

Fig. 4

Short-term effects of anoxia or hypoxia on the barley leaf. After positioning the electrodes in the substomatal cavity (pH_apo; pK_apo) and in mesophyll cells (pH_c; E_m), N₂ was flushed across the leaves, as shown in Fig. 1B, while the O₂ concentration was constantly monitored (not given). (A) Apoplastic and cytoplasmic pH; (B) membrane potential and apoplastic K⁺ (measured as pK). Traces are each representative of at least five experiments (A), and three experiments (B).

Fig. 5

Apoplastic pH of barley leaves responding to (A) N₂, flushed across a barley leaf followed by ‘light off’ (in the presence of N₂ ) or (B) to 5 mm NaCN, followed by N₂-flushing (in the presence of cyanide). The kinetics are representative of three tests each.

Fig. 6

Apoplastic pH of a barley leaf responding to treatment at the root, as indicated. Flooding (lower curve): roots of an intact plant (adapted to a moist atmosphere) were rapidly flooded with a solution consisting of 1 mm KCl, 0·1 mm CaCl₂, weakly buffered with 1 mm Mes/Tris to pH 6. After almost 2 h the reference solution was removed by suction and replaced by 10 mm NaCN + 1 mM SHAM (dissolved in the reference solution and adjusted to pH 6). Upper curve: roots resting in a moist atmosphere, after due adjustment, were flushed with N₂, while O₂ was monitored constantly (not shown). Curves are representative examples of three equivalent experiments (N₂) and four experiments (cyanide). See Materials and Methods.