Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles

Abstract

This study shows that dehydration induces imbalanced metabolism before loss of membrane integrity in desiccation-sensitive germinated radicles. Using a photoacoustic detection system, responses of CO(2) emission and fermentation to drying were analyzed non-invasively in desiccation-tolerant and -intolerant radicles of cucumber (Cucumis sativa) and pea (Pisum sativum). Survival after drying and a membrane integrity assay showed that desiccation tolerance was present during early imbibition and lost in germinated radicles. However, tolerance could be re-induced in germinated cucumber radicles by incubation in polyethylene glycol before drying. Tolerant and polyethylene glycol (PEG)-induced tolerant radicles exhibited a much-reduced CO(2) production before dehydration compared with desiccation-sensitive radicles. This difference was maintained during dehydration. In desiccation-sensitive tissues, dehydration induced an increase in the emission of acetaldehyde and ethanol that peaked well before the loss of membrane integrity. Acetaldehyde emission from sensitive radicles was significantly reduced when dehydration occurred in 50% O(2) instead of air. Acetaldehyde/ethanol were not detected in dehydrating tolerant radicles of either species or in polyethylene glycol-induced tolerant cucumber radicles. Thus, a balance between down-regulation of metabolism during drying and O(2) availability appears to be associated with desiccation tolerance. Using Fourier transform infrared spectroscopy, acetaldehyde was found to disturb the phase behavior of phospholipid vesicles, suggesting that the products resulting from imbalanced metabolism in seeds may aggravate membrane damage induced by dehydration.

Figure 1

CO laser-based photoacoustic trace gas detector. Gases emitted from samples sealed in glass cuvettes (sc) are flushed by a carrier gas (a mixture of O₂ and N₂) at a flow rate of 2 L h⁻¹ to the PA cells. Before entering the PA cells, water vapor is removed from the gas flow by a Peltier element (P) and a cold trap (D). The cold trap consists of a liquid N₂ container in which three temperatures can be chosen to condense water vapor (−5°C), water vapor and ethanol (−50°C to −65°C), or acetaldehyde (−120°C). The PA cells consist of an acoustic resonator (res) to which a microphone (mic) is attached. The IR frequencies provided by the CO laser (mixture of CO, O₂, N₂, and He) are selected using a grating device and mirrors. c, Chopper; m, mirror; pm, power meter; rc, reference cuvette; sc, sample cuvette; v, valve.

Figure 2

Example of the four-step calculation using curve fitting to determine the rates of acetaldehyde evolving from dehydrating radicles as a function of water content. Examples were taken from dehydrating pea radicles isolated after 24 h of imbibition (desiccation-tolerant stage [A–D]) and 72 h of imbibition (desiccation-intolerant stage [E–H]). Step 1 (A and E): Plots of acetaldehyde release rates are shown as a function of measuring time in the presence of the sample (●); also shown are release rates before adding the sample and after the sample moisture content had reached 0.07 g/g (○). Infrequently, high values of acetaldehyde were found before adding the samples (panels 1). They were attributed to contamination from previous experiments, which was flushed away before adding the samples. The curve is the fit to the open symbols and corresponds to the baseline. Step 2 (B and F): After subtracting the fitted curve (shown in step 1) from the data, the acetaldehyde release rates as a function of time of drying were obtained. The arrows (F) mark the data points corresponding to the onset and end point of the upsurge of acetaldehyde release rates in desiccation-intolerant radicles. A new curve was fitted to the data points excluding those in between the arrows. The fit corresponds to an exponential decay (see “Materials and Methods”). Step 3: The exponential decay obtained in step 2 was subtracted from the data points including those in between the arrows. As a result, an acetaldehyde peak can be observed in desiccation-intolerant tissues (G) but not in desiccation-tolerant radicles (C). Step 4 (D and H): The times of drying were converted to moisture content using a quadratic equation fitting the relation between drying time and moisture content (not shown) determined in parallel to the PA measurements.

Figure 3

Effect of drying on desiccation tolerance and membrane damage in radicles of cucumber (A and B) and pea (C). A, Seeds of cucumber (imbibed for 72 h) were sorted on the basis of the length of their radicle, and then dried immediately (●) or incubated in −1.5 MPa PEG for 7 d before drying (○). Subsequently, seeds were allowed to imbibe on filter paper, and desiccation tolerance was scored as a percentage of growing radicles. B and C, The effect of drying on the plasma membrane permeability of isolated radicles of cucumber (B) and pea (C) at different stages of germination: in cucumber; 42-h-imbibed (desiccation-tolerant, ○) and 72-h-imbibed radicles of 2 mm in length (desiccation-intolerant, ●), and 72-h-imbibed radicles in which desiccation tolerance had been re-induced following a PEG treatment (▵); in pea, 24-h-imbibed (desiccation-tolerant, ○) and 72-h-imbibed (desiccation-intolerant, ●) radicles. Membrane permeability was determined by ESR spectroscopy using Tempone and Tempo as spin probes introduced into the cytoplasm of cucumber and pea, respectively. The intercept between the two regression lines indicates the critical moisture content for membrane damage.

Figure 4

CO₂ production rates in imbibed radicles of pea (A) and cucumber (B) as a function of water content during drying of desiccation-tolerant (○), desiccation-intolerant (●), and PEG-induced desiccation-tolerant (▵) radicles. For each treatment, data from two representative experiments are shown together. The inset gives details of data points at a moisture content below 1.2 g/g.

Figure 5

The effect of drying on the upsurge in the acetaldehyde release rates in imbibed radicles of pea (A) and cucumber (B). In pea, desiccation-intolerant tissues were dried under air (●) or 50% O₂ (▴), whereas desiccation-tolerant radicles (○) were dried under air. In cucumber, all experiments were performed in air using desiccation-intolerant (●), desiccation-tolerant (○) and PEG-induced desiccation-tolerant (▵) radicles. For each treatment, one representative experiment is shown. The arrows indicate the critical moisture content corresponding to the onset of membrane damage.

Figure 6

The effect of drying on the ethanol release rates from imbibed radicles of pea at the desiccation-tolerant (○) and desiccation-intolerant stages (●). Data were plotted as a function of time of drying (A) and water content (B). The dashed lines and the arrow in panel A mark the lag in the time between the start of the drying experiment (and thereby ethanol emission by the tissues) and the ethanol detection by the PA cells. The lag time was always present during our measurements. It was clearly visible in dehydrating desiccation-tolerant tissues. The reason for the lag time was not investigated. In B, the data on desiccation-intolerant radicles (●) are shown only to demonstrate the occurrence of the ethanol upsurge at a moisture content similar to that of acetaldehyde (Fig. 5A). The moisture contents were obtained as described in Figure 2.

Figure 7

Plots of vibrational frequencies of the CH₂ stretch versus temperature of POPC vesicles in the absence or presence of various amounts of acetaldehyde before (open symbols) and after drying at 3% RH for 3 h (closed symbols). The arrows indicate the T_m of dried samples.