Journal of Experimental Botany, Vol. 52, No. 358, pp. 919-931,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Viability loss of neem (Azadirachta indica) seeds associated with membrane phase behaviour
1 Centre National de Semences Forestières, BP 2682, Ouagadougou, Burkina Faso
2 Department of Plant Sciences, Wageningen University, Laboratory of Plant Physiology, Arboretumlaan, 6703 BD Wageningen, The Netherlands
3 Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow, 127276, Russia
4 Department of Plant Sciences, Wageningen University, Laboratory of Experimental Plant Morphology and Cell Biology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
Received 26 May 2000; Accepted 27 November 2000
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Abstract |
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Storage of neem (Azadirachta indica) seeds is difficult because of their sensitivity to chilling stress at moisture contents (MC)
10% or imbibitional stress below 10% MC. The hypothesis was tested that an elevated gel-to-liquid crystalline phase transition temperature (Tm) of membranes is responsible for this storage behaviour. To this end a spin probe technique, Fourier transform infrared microspectroscopy, and electron microscopy were used. The in situ Tm of hydrated membranes was between 10 °C and 15 °C, coinciding with the critical minimum temperature for germination. During storage, viability of fresh embryos was lost within two weeks at 5 °C, but remained high at 25 °C. The loss of viability coincided with an increased leakage of K+ from the embryos upon imbibition and with an increased proportion of cells with injured plasma membranes. Freeze–fracture replicas of plasma membranes from chilled, hydrated axes showed lateral phase separation and signs of the inverted hexagonal phase. Dehydrated embryos were sensitive to soaking in water, particularly at low temperatures, but fresh embryos were not. After soaking dry embryos at 5 °C (4 h) plus 1 d of further incubation at 25 °C, the axis cells were structurally disorganized and did not become turgid. In contrast, cells had a healthy appearance and were turgid after soaking at 35 °C. Imbibitional stress was associated with the loss of plasma membrane integrity in a limited number of cells, which expanded during further incubation of the embryos at 25 °C. It is suggested that the injuries brought about by storage or imbibition at sub-optimal temperatures in tropical seeds whose membranes have a high intrinsic Tm (10–15 °C), are caused by gel phase formation. Key words: Azadirachta indica, chilling stress, imbibitional stress, membranes, seed storage.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Seeds that can be dried to a moisture content (MC) of 5% are generally regarded as desiccation tolerant. They can usually be stored for periods of many years. However, some seeds, often of tropical origin, cannot withstand dehydration. They are also often chilling sensitive (Corbineau and Côme, 1988
; Chin et al., 1989; Tompsett, 1994). As a result, such seeds cannot be preserved for long periods of time (Roberts, 1973). A further group of seeds that demonstrate behaviour intermediate between the afore-mentioned categories of desiccation tolerance and storage behaviour has been described (Ellis et al., 1990, 1991). When they originate from tropical climates, such intermediate seeds are often chilling sensitive.
Neem (Azadirachta indica) is an important tropical tree species with many uses, whose seeds have been categorized as having intermediate storage longevity (Sacandé et al., 1996, 1998; Hong and Ellis, 1998). Neem seeds are chilling sensitive at MC 10%. Their limited desiccation tolerance has been partially attributed to their sensitivity to imbibitional stress below 10% MC. Survival after dehydration was improved by rehydrating the dry seeds at elevated temperatures of around 35 °C (Sacandé et al., 1998). This sensitivity to both chilling and imbibitional stress has contributed to neem seed's reputation as being difficult to store.
Many plants of tropical origin suffer injury when they are kept below 10–15 °C for some time. Apart from the dysfunction of some membrane enzymes (Lyons et al., 1979a, b; Yamawaki et al., 1983; Yoshida et al., 1986), increased leakage of cytoplasmic solutes from the cells has been observed (Bergevin et al., 1993; Bertin et al., 1996). This chilling sensitivity has been attributed to a phase transition in cell membranes from the liquid crystalline to the gel phase (Lyons et al., 1979a, b; Wang, 1982), often followed by lateral phase separation, i.e. the sorting of membrane components according to their molecular species (Platt-Aloia and Thomson, 1987; Sharom et al., 1994). In intact tomato hypocotyls, for example, the phase transition temperature (Tm) of the membranes as measured by Fourier transform infrared (FTIR) spectroscopy was around 10–15 °C (Crowe et al., 1989b). In contrast, the in situ Tm values of plants from temperate zones are usually around or below 0 °C (Crowe et al., 1989b; Hoekstra et al., 1991).
During imbibition (rehydration), dry seeds leak intracellular solutes into the surrounding medium. If the leakage persists, it is a sign that plasma membranes are damaged. The causes of imbibitional leakage have been extensively studied in pollens and model lipid systems (Crowe et al., 1989a; Hoekstra et al., 1992). The Tm of membranes increases during dehydration, and the gel phase may thus be formed at ambient conditions, depending on the phospholipid composition (reviewed in Hoekstra and Golovina, 1999). Imbibitional damage has been attributed to plasma membranes being in the gel phase at imbibition (Crowe et al., 1989a). Melting these membranes prior to imbibition by preheating or prehydration from the vapour phase can alleviate the stress (Hoekstra and Van der Wal, 1988). It has recently been hypothesized that the rigidity of the plasma membrane at imbibition is the critical factor determining whether the organ(ism) survives rehydration or not (Hoekstra et al., 1999). Increasing the elasticity of the plasma membranes would prevent rupture during imbibition.
It has also been suggested that the sensitivity to chilling of neem seed is associated with a possibly high Tm of plasma membranes in the fresh seed (Sacandé et al., 1998). Hence, lowering the temperature below Tm would lead to gel phase formation and lateral phase separation, followed by leakage. In the same way, the extreme sensitivity to imbibitional stress of dry neem seed has been hypothesized to depend on such an intrinsically high Tm of the plasma membranes (Sacandé et al., 1998). The presence of gel phase just before imbibition would lead to permanent damage during rehydration.
In this paper, these hypotheses were tested. Membrane barrier properties were investigated using a nitroxyl spin probe technique. This technique has been used to study the in situ membrane integrity and permeability of very small amounts of plant material (Smirnov et al., 1992; Golovina et al., 1997). Electron microscopy was used to investigate changes in membrane structure associated with chilling and imbibitional damage. The in situ Tm of membranes was determined by FTIR microspectroscopy. The behaviour of membranes in this tropical tree seed species is discussed in the light of the intrinsically high Tm, and directions for improving protocols for the storage of tropical seeds with difficult storage behaviour are given.
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Materials and methods |
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Plant material
Experiments were carried out using seeds from yellow fruits of neem (Azadirachta indica A. Juss.), which were harvested from plantations at Ouagadougou, Burkina Faso. The seeds were prepared using the method described previously (Sacandé et al., 1996
, 1997). The seed covers (endocarp) of the fresh seeds were removed to utilize embryos for the determination of initial water content and germination capacity. Three replicates of five embryos were weighed, then dried at 96 °C for 48 h and again weighed to determine the MC calculated as a percentage of the fresh weight (FW) (for tropical seeds, ISTA, 1993).
Determination of germination
The embryos were soaked in tap-water for 4 h at different temperatures (2–40 °C) and thereafter transferred to a constant temperature of 25 °C for germination on wetted filter paper in Petri dishes (10 cm diameter). Other embryos were incubated directly in the Petri dishes for germination at a range of different temperatures. Replicates of 10–15 embryos per Petri dish were used for the determination of germinability. The seeds were inspected regularly for 4 weeks, and the germinated individuals were removed. Embryos were considered as germinated when radicles had elongated by 2–3 cm (for tropical seeds, ISTA, 1993). Statistical analysis of the germination data was carried out using the 
2-test, and differences were regarded as significant at P
0.05.
Measurements of K+-leakage
The level of K+ was measured in the eluate of embryos, using a flame photometer (PFP 7, Jenway, Felsted, UK). Fresh embryos which had been maintained in humid conditions at 5 °C or 25 °C for different durations (up to 18 d) were used. After weighing, 10 such embryos were incubated in 40 ml of Milli-Q (Millipore) water in Petri dishes at 25 °C, and the K+ leached from the embryos was regularly determined in 4 ml of eluate. K+-leakage from the embryos was linear with time over the first 30 h of incubation at 25 °C and was expressed as µmol h-1 g-1 FW.
Electron paramagnetic resonance (EPR) spin probe measurements
A nitroxyl spin probe technique was used to estimate the integrity of plasma membranes in neem seed tissues. EPR spectra were obtained at room temperature using an X-band EPR spectrometer (model 300E, Bruker Analytik, Rheinstetten, Germany). Embryonic axes and cotyledon cubes from fresh neem seeds were excised after different times of storage or imbibition. Samples were put into capillaries (2 mm diameter), then 1–2 µl of a solution containing 1 or 2 mM perdeuterated TEMPONE (4-oxo-2,2,6,6-tetra-methyl-1-piperidinyloxy) and 120 mM potassium ferricyanide was added. Spectra were recorded at a power of 2 mW (20 dB) and a modulation amplitude of 0.5 Gauss, conditions that excluded saturation and overmodulation of the EPR spectra. They were normalized to the same concentration of TEMPONE and the same number of scans, so as to be able to compare spectra. Each sample contained approximately the same amount of cotyledon tissue or one axis.
The EPR spin probe technique is based on the differential permeability of membranes to the stable TEMPONE radical and to ferricyanide ions that broaden the signal. Adding TEMPONE to cells results in an EPR spectrum that is the sum of signals from all environments where TEMPONE is present. To selectively remove the extracellular spin probe signal, plasma membrane-impermeable ferricyanide ions were added. Within the cell, TEMPONE molecules partition between the lipid fraction (oil and membranes) and the aqueous cytoplasm. The intracellular EPR signal from TEMPONE consequently originates from two different surroundings—lipid and water—which are resolved in the high field (right-side peaks) region of the spectrum. When ferricyanide penetrates the cytoplasm through deteriorated plasma membranes, the aqueous signal disappears, but the lipid signal remains because the lipid phase is impermeable to ferricyanide. The amplitude of the aqueous signal was used to estimate membrane integrity.
Transmission electron microscopy (TEM)
Samples from seed tissues were glued with TBS (Electron Microscopy Sciences, Washington, USA) onto golden specimen carriers (3 mm, Bal-Tec AG, Liechtenstein) that were placed on a polished brass block cooled by melting ice. Immediately after fixing the specimens, the carriers were plunge-frozen in liquid propane at -180 °C and stored in liquid nitrogen (-196 °C). Freeze-fracture replicas were prepared in a BAF 400 freeze–fracture apparatus (Bal-Tec AG, Liechtenstein) at -120 °C and 10-7 Torr. Platinum was evaporated at an angle of 40°, and carbon was evaporated to support the replicas. The replicas were immersed in 60% glycerol to alleviate cracking, then cleaned with a 50% CrO3 solution for 4 h and soaked overnight in bleach solution. The replicas were rinsed in distilled water and collected on EM grids. Electron micrographs were made with a TEM (JEM-1200 EX II, Jeol, Japan) at 80 kV. The membrane fracture face nomenclature of Branton et al. was used for the description of the freeze-fracture images (Branton et al., 1975).
Low temperature scanning electron microscopy (LTSEM)
Whole axes and cotyledon tissue (from 3 seeds for each treatment) were fixed onto a stub with colloidal carbon adhesive (LeitC Neubauer Chemikalien, Münster, Germany). The stubs were placed on a specimen holder and immediately plunged into LN2 (-196 °C). The frozen samples were transferred into a cryo-transfer unit (CT 1500-HF Oxford Instruments, UK). The unit consisted of a cryo-preparation chamber at high vacuum (10-6 Pa) dedicated to an LTSEM (JEOL, model 6300 F, Japan) and a cryo-stage inside the microscope. The specimens were placed inside the cryo-chamber at -85 °C, immediately freeze-fractured with a cold scalpel, and freeze-dried for 2 min at -85 °C and 10-7 Torr. The samples were then sputter-coated with 10 nm platinum. The LTSEM was used to examine the coated seed tissue at 5–10 kV, keeping the temperature of the specimens at -180 °C.
Determination of the Tm of membranes by FTIR
In situ FTIR microspectroscopy was used to determine the Tm of membranes in hydrated 10-d-old seedling roots. FTIR spectra were recorded on a Perkin-Elmer 1725 IR-spectrometer (Perkin-Elmer, Beaconsfield, Buckinghamshire, UK), equipped with a liquid nitrogen-cooled mercury/cadmium/telluride detector and a Perkin-Elmer microscope using the method described earlier (Wolkers and Hoekstra, 1995). A thin slice from the root tip was rapidly transferred to a cooled sample holder. The slice was placed between two CaF2 windows that were tightly mounted into a temperature-controlled brass cell. The temperature of the sample in the instrument was regulated with a computer-controlled device that activated a liquid nitrogen pump, in conjunction with a power supply for heating of the cell. The sample temperature was recorded using two PT-100 elements that were located close to the sample windows. The temperature dependence of the FTIR spectra was studied in the range between -60 °C and 70 °C, starting with the lowest temperature. Spectra were recorded every minute at temperature increments of 1.5 °C min-1. The instrument was purged of water vapour with a Balston dry air generator (Balston, Maidstone, Kent, UK).
The spectral region between 3000 and 2800 cm-1 was selected and second derivative spectra were calculated (19 points smoothing factor; calculated using the Infrared Data Manager Analytical Software, version 3.5 [Perkin Elmer]). The position of the symmetric CH2 stretching vibration band of the hydrated membranes around 2852 cm-1 was determined from these second derivative spectra.
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Results |
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Chilling stress in hydrated fresh seeds
Effect of temperature on germination
When highly viable (96% germinative capacity) fresh (37.8% MC) seeds were incubated in water at a range of constant temperatures, none germinated at
10 °C (Fig. 1A). Maximum germination occurred when seeds were incubated at 15 °C, indicating that the threshold minimum temperature for germination lies between 10 °C and 15 °C. However, embryos took 2 weeks to complete their germination at 15 °C (Fig. 1B), whereas incubation at 25 °C or 30 °C reduced this period to 6 d and 4 d, respectively. Final germination percentages obtained at temperatures ranging from 15–30 °C did not differ significantly from each other.
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Storage at chilling temperatures: effects on germination and K+-leakage
The leakage of K+-ions from fresh embryos kept at 5 °C or 25 °C was measured, so as to investigate the effect of storage at low temperature on the barrier properties of the plasma membranes. Embryos kept at 25 °C showed high germination over the entire storage period of 18 d, and leakage rates remained low (Fig. 2). Storage at 5 °C led to a gradual increase in leakage rate, which coincided with a gradual decrease in germination percentage. None of these chilled embryos germinated after 14 d of storage at 5 °C.
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Storage at chilling temperatures: effect on membrane integrity
Since it is impossible to measure K+-leakage from the small neem axes (0.3 mg dry matter) with flame photometry, membrane integrity was studied with the more sensitive spin probe technique. A selection of typical EPR spectra of TEMPONE from embryonic axes during storage of fresh embryos at 5 °C is shown in Fig. 3. The spectra contained cytoplasmic and lipid components, discernible at the high field (right side) region of the spectrum. Spectra were recorded from approximately similar amounts of material, because each sample contained one single axis of approximately similar size. The amplitude of the aqueous peak therefore correlates with the proportion of living cells in an axis. The amplitude of the cytoplasmic component decreased with time of cold storage at 5 °C, indicating a gradual decrease in the proportion of living cells with intact membranes that are impermeable to ferricyanide. During the 25 °C storage (control), the aqueous peaks did not change much (spectra not shown).
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The proportion of living cells in the axes, expressed as the average amplitude of the cytoplasmic component, decreased during cold storage at 5 °C (Fig. 4
). However, the amplitude did not change during the first 4 d, although the germination capacity (Fig. 2
) was already reduced. It may be that the extent of membrane damage after 4 d of cold storage was still too small to be detected by the EPR spin probe technique. However, the injuries could have amplified in the germination test during the incubation in the Petri dishes at 25 °C. To test this possibility, EPR spectra of TEMPONE from axes after several days of incubation of the 4 d chilled embryos in the Petri dishes at 25 °C were also recorded. Only non-germinating embryos were selected for this analysis of plasma membrane integrity. The amplitude of the aqueous peaks decreased from 205±26 (±SE; n=6) for the 4 d chilled axes without further incubation at 25 °C to 125±13 after 2 d of incubation, and to 88±53 after 5 d. This decrease in amplitude with days of incubation might be interpreted to mean that some slight, unnoticeable membrane damage that existed already after 4 d of cold storage, developed further during incubation at 25 °C, which may explain the failure of the embryos to germinate.
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Cotyledons were also probed for membrane intactness with the spin probe technique. The spectrum of TEMPONE in fresh neem seeds (viable) before storage at 5 °C was characterized by a large cytoplasmic component and a smaller but well pronounced lipid component (control in Fig. 5
). The EPR spectra from cotyledons of embryos with axes damaged by cold storage varied considerably, even within parts of the same cotyledon. A selection of these spectra obtained from cotyledons (two different locations) of three individual embryos after 14 d of storage at 5 °C is shown in Fig. 5.
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The EPR spectra from embryo 1 (both samples) and from embryo 2 (sample 1) had a shape similar to those from the control sample (0 d of cold storage), representing viable tissue. In contrast, spectrum 2 from embryo 2 had a considerably reduced cytoplasmic component, and both spectra from embryo 3 lacked the cytoplasmic component, indicative of dramatic membrane disruption (tissues non-viable). Apparently, damage in cotyledons does not occur simultaneously in all cells. These data are supported by the results of tetrazolium staining, which showed patches which were unstained among stained tissue (data not shown).
The EPR spectra from cotyledon tissue in embryos after 4 d of storage at 5 °C did not show any damage (spectra not shown) and resembled those from the control sample in Fig. 5, characterizing viable tissue. It was found that also in cotyledon tissue damage appeared to develop during subsequent incubation for germination at 25 °C as in the case of axes (spectra not shown).
Storage at chilling temperatures: effect on plasma membrane ultra-structure
The possible causes of leakage and increased permeability of the plasma membranes of fresh embryos stored at 5 °C were studied using freeze-fracture and TEM. Figure 6A shows a representative freeze-fracture image of the protoplasmic face (PF) side of a plasma membrane in a fresh neem embryonic axis, displaying randomly distributed intra-membrane particles (IMP). Some plasmodesmata (white arrows) are clearly visible. After 14 d of storage of fresh embryos at 5 °C when the membrane damage was evident (Figs 2–5), a number of features can be observed in the axes (Fig. 6B–D). Figure 6B shows an image of a replica of the PF side of a plasma membrane having a reasonably random distribution of IMP, with the imprints of cellulose fibrils from the cell wall clearly visible. Patterns of phase separation are evident in Fig. 6C, with the IMP accumulated in particular areas of the replica and IMP-free zones in between. The image shows a depression that resembles a fracture jump lesion, similar to the lesions observed in freeze-injured rye leaves (Webb and Steponkus, 1993). A more detailed inspection of this replica (Fig. 6C) revealed bands of tubular structures that were preliminarily identified as inverted hexagonal phase (HII) (Fig. 6D).
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Phase transition of membranes in situ
In an attempt to understand the mechanisms involved in the sensitivity to chilling, the in situ Tm of membranes was probed using FTIR microspectroscopy. By following the wave number of the symmetric CH2 stretching vibration band (at 2852 cm-1) during heating of a sample, information can be obtained about the relative packing density of the acyl chains of lipids (Casal and Mantsch, 1984). However, the abundant oil in fresh seeds and, even in the axes, confounded the spectra too much to be able to determine thermal events attributable to membranes. Roots of germinating 10-d-old seedlings which had consumed most of their oil, were therefore utilized as the experimental material. Figure 7 shows a representative wave number versus temperature plot for a slice of an axial root tip. The mid-value during the co-operative phase transition from the gel to liquid crystalline phase lies at approximately 9.6 °C. To verify that oil did not contribute too much to this plot, a wave number versus temperature plot of oil pressed out of a non-germinated axis is also presented. From this plot a Tm of -4.8 °C was derived. Even if the root tip slice had residual amounts of oil, it is clear that the estimated Tm of the lipids in the root tip is higher than that of pure oil, suggesting that there was a considerable contribution of membrane lipids to the spectrum. If correction for traces of neutral lipid in the root tip plot were possible, it would probably increase the estimated Tm of membranes by at most a few °C. This value nevertheless demonstrates that the Tm of membranes in fresh neem tissue is in the range of what has been found for other tropical plant species.
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Imbibitional chilling stress in dry neem seeds
Effect of imbibition temperature on germination
With dehydration, neem seeds become increasingly sensitive to rehydration in water. Imbibitional damage is more extensive at lower soaking temperatures and increases with the age of the dry seeds (Sacandé et al., 1998). To investigate the sensitivity to imbibition, embryos (seeds without endocarp) from a fresh (37.8% MC) and a dried (4.3% MC; after 6 weeks at 32% RH, 20 °C) neem seed lot were soaked for 4 h in water at a range of temperatures, followed by incubation in water (Petri dishes) at 25 °C. Figure 8 shows the percentage of germinated embryos after these treatments. More than 90% of the fresh embryos germinated at all of the imbibition temperatures tested, indicating that the fresh seeds were not sensitive to soaking under chilling conditions. In contrast, germination of the dry embryos was low at low soaking temperatures and high at elevated soaking temperatures, 30–40 °C being optimal.
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Imbibitional stress: effect on plasma membrane integrity
To investigate the relationship between imbibitional injury and plasma membrane damage, EPR spectra of TEMPONE were recorded from axes in dry embryos directly after the 4 h soaking at 5 °C or 35 °C, and after the next 1–3 d of incubation at 25 °C. Soaking at 5 °C killed the embryos, whereas embryos soaked at 35 °C germinated well. The amplitudes of the aqueous cytoplasmic component remained high and constant during the entire period after the 35 °C treatment (Fig. 9). In contrast, those after the 5 °C treatment were lower, particularly on the second and third day after the treatment. When considering the amplitude of the aqueous cytoplasmic component to be a measure of membrane integrity, then the proportion of cells with disrupted membranes progressively increased with time after the soaking at 5 °C and was low throughout after the soaking at 35 °C.
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Although the lower amplitude of the cytoplasmic component in axes just after the 4 h of soaking at 5 °C can be interpreted as being caused by improper swelling, it can also be due to the presence of a small number of cells with increased membrane permeability. To investigate this possibility, the shape of the high-field component was analysed. Figure 10
shows the high-field lines of the EPR spectra of TEMPONE from axes of embryos imbibed at 5 °C and 35 °C fitted for amplitude and peak position. Although there was no increase in the peak-to-peak line width—a sign that the majority of cells were still impermeable to ferricyanide ions—the wings of the lines were more broad in the spectrum of the sample after soaking at 5 °C. This can be caused by an unresolved contribution to the spectrum from a small proportion of cells that has higher permeability to ferricyanide ions and, consequently, increased peak-to-peak width (Sankaram and Marsh, 1998). Such broadened wings were observed in all the samples imbibed at low temperature, and not in those soaked at 35 °C. The wings are interpreted as being associated with a constant population of dying cells having increased membrane permeability.
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LTSEM inspection of imbibitional injury
The occurrence of injured plasma membranes after imbibition at 5 °C was further supported by LTSEM images of the axis cells after 1 d of incubation at 25 °C following the soaking treatment. Figure 11 shows representative LTSEM images from axes after the two treatments. Whereas after the 35 °C-soaking treatment plus 1 d of incubation at 25 °C (Fig. 11A) the axis cells had a surface morphology typical of swollen, turgescent cells, those after the 5 °C soaking treatment were not turgescent and appeared wrinkled (Fig. 11B).
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Cryofracturing revealed the internal structure of the axis cells. In the 35 °C-soaked axis cells the fracture often occurred across the core of the plasma membranes (Fig. 11C
), but almost never in the 5 °C-soaked axes (Fig. 11D). The cytoplasm appeared to be appressed to the plasma membrane after the 35 °C-soaking treatment, and vacuoles were apparent. Cryo-fracture images of the 5 °C-soaked axis cells show that there was space between the cell wall and the remainder of the plasma membrane enclosing the cytoplasm, which supports the suggestion that these cells were not turgescent. This lack of turgescence must be ascribed to reduced barrier properties of the plasma membrane after the soaking treatment followed by 1 d of further water uptake at 25 °C. Figure 11E and F show details of the plasma membranes of axis cells after the 35 °C treatment, with pits and plasmodesmata clearly visible (E, F), and imprints of cellulose microfibrils (F) on the exoplasmic face (EF). This indicates the firm appression of the plasma membrane to the cell wall, which is typical of a turgescent cell.
A similar picture emerged when the same LTSEM inspection of cotyledons was performed, except that the surface morphology of the 35 °C and 5 °C treatments appeared rather similar (Fig. 12A, B). As for the axes, cryofracturing caused the fracture plane to tend to follow the core of the plasma membranes after the 35 °C treatment (Fig. 12C), but not after the 5 °C treatment (Fig. 12D). The cytoplasm appeared appressed to the plasma membrane after the 35 °C-soaking treatment, with organelles and oil bodies clearly visible. Cryo-images of the 5 °C-soaked cotyledon cells show a space between the cytoplasm and the cell wall, and the cytoplasm has a disorganized appearance lacking clearly discernible organelles. The cell walls had an undulating appearance, indicating loss of turgor. Figure 12E and F show details of Fig. 12C and D, respectively. The intercellular space between cotyledon cells after the 35 °C treatment had a triangular shape and no water, in contrast to that after the 5 °C treatment, which had a spherical shape and was filled with water.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Chilling stress
The viability of seeds of tropical origin is known to be adversely affected by chilling temperatures (Corbineau and Côme, 1988
; Chin et al., 1989; Tompsett, 1994). It has been shown that stored neem seeds are sensitive to chilling temperatures when the MC of the seed is 10% (Sacandé et al., 1998). This sensitivity to chilling limits the potential for the long-term maintenance of neem seed viability during storage. It was hypothesized that the causes of chilling sensitivity may lie in the elevated Tm of membranes in neem seeds, as it is for vegetative tissues of a few other tropical plants (Tm ranging from 10–15 °C; Crowe et al., 1989b; Mazliak, 1992). Although it was difficult to determine Tm in seeds because of the abundant oil, FTIR spectra of fresh root tips of neem seedlings indicate a Tm of at least 9.6 °C (Fig. 7), and probably a few °C higher. This implies that below about 10 °C, membranes tend to be in the rigid gel phase. As a result, structural changes could develop that lead to leakage of solutes from hydrated seeds. It was indeed found that fresh embryos leaked more K+ the longer they were stored at 5 °C, and that this coincided with a decrease in germination capacity (Fig. 2). The spin probe method provided further evidence for reduced integrity of the plasma membranes in axes with time at 5 °C (Figs 3, 4, 5). However, directly after the 4 d of cold storage, the data on both the leakage (Fig. 2) and plasma membrane integrity (Fig. 4) could not predict the 50% reduction in germinative capacity. During the lag time of germination at 25 °C, an undetectable primary damage could have led to the loss of plasma membrane integrity as a secondary effect. In addition, injury to other membrane systems such as mitochondria and, consequently, metabolic disruption of the cells as a secondary effect, could have caused the progress in the loss of plasma membrane integrity and the failure of the embryos to germinate.
A study of the plasma membrane ultrastructure supports the view that a phase transition occurs below 10 °C in fresh neem seed (Fig. 6). After 14 d at 5 °C, IMP-free areas were found in the plasma membranes of axes adjacent to areas of clustered IMP (Fig. 6C), although intact cells with random distribution were also apparent (Fig. 6B). The areas of clustered IMP point to lateral phase separation. HII-like structures were observed, which may have formed upon phase separation. It is not entirely clear whether the lateral phase separation and HII phase are primarily a result of the low temperature or whether they are a secondary effect of leaky membranes in the deteriorating cells. Besides the occurrence of the HII phase, an interesting feature that was observed is the fracture jump lesion in the replica of the plasma membrane of the chilled seed (Fig. 6C). In freezing damaged rye leaves, this lesion was ascribed to freeze-induced dehydration (Webb and Steponkus, 1993). Here, no such dehydration took place because the temperature was not reduced below 0 °C, so the appearance of this type of lesion is attributed to the effects of the transition of the plasma membrane to the gel phase. In rye, the temperature probably has to be lowered to far below 0 °C to evoke a phase transition to the gel phase in the plasma membranes—a temperature that also allows the formation of ice crystals.
There are indications that further dehydration of neem seeds below 10% MC alleviates chilling/subzero-°C stress during storage (Hong and Ellis, 1998; Sacandé et al., 1998, 2000). This may be due to the absence of lateral phase separation, probably because of a restricted lateral mobility of the membrane components. It is likely that this mobility is curtailed at low water contents because of hydrogen bonding interactions with the glassy cytoplasmic matrix (Sacandé et al., 2000).
Fresh neem embryos did not germinate when incubated at temperatures of 10 °C (Fig. 1). The minimum temperature for germination thus appears to be between 10 °C and 15 °C. Although not examined for membrane damage, it is expected that similar membrane phase behaviour is involved in the inability of the embryos to germinate.
Imbibitional stress
A number of seeds, particularly those of tropical origin, are damaged when they are plunged into water at low temperatures (Hobbs and Obendorf, 1972; Cohn and Obendorf, 1976; Woodstock et al., 1985). The seeds of cotton, soybean, cowpea, maize, and sorghum are typical examples. These phenomena have also been observed in pollen and yeast (Van Steveninck and Ledeboer, 1974; Hoekstra, 1984). The decrease in germinability coincides with increased leakage of solutes from the tissue. The injury can be prevented by prehydration from the vapour phase or preheating before imbibition in water or germination medium (Cal and Obendorf, 1972; Hoekstra and Bruinsma, 1975; Hoekstra, 1984; Hoekstra and Van der Wal, 1988). It is thought that the phase of the plasma membranes just before imbibition is involved in imbibitional injury (Crowe et al., 1989a). It has recently been hypothesized that the mechanical properties of the plasma membranes determine the ability to withstand imbibition (Hoekstra et al., 1999). Plasma membranes of pollen exhibited holes within seconds after imbibition when they were in gel phase just before imbibition. The treatments that alleviate imbibitional stress such as prehydration from the vapour phase and preheating also melt the plasma membranes.
Because of the intrinsically high Tm of membranes in tropical plants and the fact that dehydration tends to increase this Tm, it seems logical that tropical seeds are particularly sensitive to imbibitional damage. In Fig. 7 it is shown that neem has a membrane Tm typical of tropical plants. Previously it was demonstrated that neem seeds become sensitive to imbibitional stress after drying, a phenomenon that is augmented during ageing under dry conditions (Sacandé et al., 1998). In addition, in Fig. 8 it is shown that dry embryos are sensitive to soaking in water during the first 4 h, particularly at chilling temperatures. Hydrated embryos were insensitive to the 4 h soaking at low temperatures, which apparently was too short to cause problems in the plasma membranes.
Support for a possible involvement of plasma membranes in the imbibitional injury comes from the results of the spin probe experiments. The amplitude of the aqueous signal decreased with time after the 4 h imbibition at 5 °C (Fig. 9). Although not evident directly after the imbibitional chilling treatment on the basis of amplitude, damage in some cells was evident on the basis of the line shape of the spectra (Fig. 10). It is suggested that many axis cells may still be alive just after the cold imbibition. In contrast to relatively simple systems such as pollen and yeast in which plasma membrane damage occurs immediately upon the stress (Hoekstra et al., 1999), the damage to the multicellular embryos may be initially limited to some outer cell layers (Powell and Matthews, 1978). Subsequently, the damage spreads with time during incubation at 25 °C. All cells that were still alive, first pass through a stage of increased membrane permeability and then die. Further support for a role of the plasma membranes is provided by the LTSEM micrographs depicting axes and cotyledons after soaking of dry embryos at 5 °C and 35 °C followed by 1 d incubation at 25 °C (Figs 11, 12). The surface morphology of the axes clearly indicates loss of turgor after soaking at 5 °C, in contrast to the swollen appearance of cells after soaking at 35 °C. The ultrastructure of the cryo-fractured cells supports this view. The strong appression of the plasma membrane to the cell wall, as evidenced by the imprints of cellulose microfibrils after the 35 °C imbibition treatment, is proof of turgescent, viable cells. The 1 d incubation following the 4 h soaking was meant to allow the embryo to rehydrate fully. In the case of soaking injury it also allowed further deterioration of the cells. The axis and cotyledon cells of the 5 °C soaking treatment did indeed have a fully deteriorated appearance. The fracturing did not follow the core of the plasma membranes, which can be interpreted to mean that these membranes had changed physical properties. Thus, also in seeds, damage to the plasma membranes is suggested to underlie imbibitional chilling injury.
Dry seeds of papaya, characterized as exhibiting intermediate storage behaviour (Ellis et al., 1991), fail to germinate when they are incubated in water at 20 °C, but do so at higher temperatures (Wood et al., 2000). It has been shown that they remain viable for some time, even when they do not germinate. The seeds need an additional heat treatment to release the dormancy and allow them to germinate. This behaviour has been described as dehydration-imposed dormancy. The LTSEM micrographs (Figs 11, 12) of neem embryos indicate that the reduced germination below the optimal soaking temperatures of 30–40 °C is likely to be due to imbibitional damage and not to dehydration-imposed dormancy.
The results reported in this paper suggest that the elevated Tm of membranes in neem, a tree of tropical origin, is associated with the sensitivity of seeds to both chilling and imbibitional stress. A high Tm increases the likelihood of the occurrence of the rigid gel phase when membranes are dehydrated or chilled. Membranes in the gel phase are assumed to be responsible for the injuries caused by chilling as well as imbibition. These sensitivities may explain the conflicting reports in the literature on desiccation tolerance and storage longevity of neem seeds. Seeds may have been killed during rehydration rather than during dehydration and storage. Protocols for handling and storage of neem seed and tropical seeds in general, which pay special attention to appropriate rehydration procedures, are recommended.
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The financial support by the Dutch Organization for the Advancement of Tropical Research (WOTRO) to MS is gratefully acknowledged. This work was also supported by grant No. 047.01.006.96 from the Netherlands Organization for Scientific Research and NATO collaborative linkage grant No. LST.CLG 975082 to EAG. We would like to thank Professor I Grigoriev (Institute of Organic Chemistry of the Russian Academy of Sciences, Novosibirsk, Russia) for the kind gift of perdeuterated TEMPONE.
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5 To whom correspondence should be addressed. Fax: +31 317 484740. E-mail: folkert.hoekstra{at}algem.pf.wag\|[hyphen]\|ur.nl
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