Journal of Experimental Botany, Vol. 52, No. 358, pp. 1015-1027,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
The competence to acquire cellular desiccation tolerance is independent of seed morphological development
1 Timiryazev Institute of Plant Physiology, Botanicheskaya 35, Moscow, 127276, Russia
2 Department of Experimental Plant Sciences, Wageningen University, Laboratory of Plant Physiology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
3 Department of Experimental Plant Sciences, Wageningen University, Laboratory of Experimental Plant Morphology and Cell Biology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
Received 20 June 2000; Accepted 30 November 2000
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Abstract |
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Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.
Key words: Cellular desiccation tolerance, embryogenesis, EPR spin probe, morphogenesis, premature drying, SEM, Triticum aestivum.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In general, during seed formation four developmental programmes have been recognized, morphogenesis, maturation, dormancy, and germination, which are considered to be strongly regulated and integrated (Walbot, 1978
; Raghavan, 1986; Kermode et al., 1986; Kermode, 1990). However, some late steps in these developmental programmes, such as dormancy or storage reserve accumulation (maturation), can be bypassed without harm to the seeds, suggesting that the integration is less strong than formerly assumed (McCarty, 1995).
The acquisition of desiccation tolerance is an essential part of the maturation programme in most types of seeds. Desiccation tolerance, defined as the ability of a seed to germinate after fast drying (Bewley and Black, 1994), is acquired at the stage of mass maturity or a few days earlier, but certainly before maturation drying. This sequence of events suggests that acquisition of desiccation tolerance is a developmental process, rather than a response to maturation drying. The acquisition of desiccation tolerance involves structural and biochemical changes in the cell, such as the substitution of large vacuoles by plastids containing storage substances, the accumulation of di- and oligosaccharides, and the synthesis of dehydrins (for reviews see Bewley and Black, 1994; Dure, 1997). All of these changes occur during maturation, usually when morphogenesis is completed, from which it has been deduced that desiccation tolerance is acquired only by a morphologically developed embryo.
A large number of references indicate that slow premature drying in pods or on ears nevertheless allows the formation of viable, desiccation-tolerant seeds (Goff, 1900; Harlan and Pope, 1922, 1926; Sprague, 1936; Nutman, 1941; King, 1976; Adams and Rinne, 1981; Sanhewe and Ellis, 1996). The size and weight of such seeds depended on the time when drying was imposed. For example, during the slow drying of rye kernels on ears (with the straw attached) that were cut from the plant at 5 d after anthesis (daa), the non-differentiated 16-cell pro-embryos developed into dwarf embryos (Nutman, 1941). These dwarf embryos had a reduced number of foliage and root primordia when compared with the number in normal embryos, but this was still sufficient for germination. All the embryonic organs were proportionately reduced in size. The difference in organ size between dwarf and normal embryos was due to a difference in cell number, but the cell size in homologous organs was the same. Beside surviving dehydration, the dwarf embryos were able to germinate and to develop into normal plants. Five daa was the earliest time at which the ear could be excised without this leading to abortion of the ovary. A similar critical time was established for prematurely ear-dried barley kernels (Harlan and Pope, 1922). Soybean seeds that had been dried in the pod before 17 daa were considered non-viable (Adams and Rinne, 1981). Apparently, in such seeds the basic morphogenesis was incomplete and, consequently, the competence to form seedlings was lacking. However, the absence of germination in these very prematurely dried seeds does not necessarily mean that desiccation tolerance at the cellular level was not acquired. From the observation that developmentally arrested embryos in corn mutants did not become necrotic (Sheridan and Clark, 1993), it can be implied that desiccation tolerance is acquired at the cellular level without the appropriate morphogenesis.
To investigate whether cellular desiccation tolerance can be acquired in poorly developed embryos, the usual method of assessment, testing germination, is unsuitable. Plasma membrane integrity after drying and rehydration can be utilized as a criterion for cellular desiccation tolerance because membranes are considered a primary target of desiccation injury (Senaratna and McKersie, 1983; Senaratna et al., 1984). Leakage assays that are routinely used to test membrane integrity are inadequate in this case, because the size of the poorly developed embryos is too small to influence the overall leakage from the seed. In addition, these embryos are too small to be dissected from the surrounding tissues. Recently, a spin probe method has been applied to study the integrity of cellular membranes in seeds with ageing (Golovina et al., 1997). The high sensitivity of the method permits the study of very small samples, such as individual wheat embryos.
In the present work it was investigated whether very premature, ear-dried wheat kernels with an incomplete embryonic morphogenesis can acquire desiccation tolerance. To this end, an electron paramagnetic resonance (EPR) spin probe technique and low temperature scanning electron microscopy (LTSEM) were used. The results indicate that acquisition of desiccation tolerance at the cellular level is independent of the extent to which morphogenesis has progressed.
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Materials and methods |
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Plant material
Plants of spring wheat (Triticum aestivum cv. Priokskaya) were grown in pots under greenhouse conditions (16 h [supplemental] light/8 h dark and day/night temperatures of 22/20±2 °C). During the experiments, greenhouse conditions did not vary to an extent that they influenced the rate of development. Ears were tagged at the beginning of anthesis. Embryos were isolated from the kernels during development. Rapid drying of the embryos was performed in a flow of dry air (relative humidity [RH]=3%), low moisture contents (MCs) (7% on a fresh weight basis) being reached within 2 h. Alternatively, plants were cut at ground level at different times after anthesis and dried down under laboratory conditions in diffuse day light (approximately 23 °C and 30% RH). Threshing was done at least one month later. Kernels were removed from the ear and allowed to imbibe on filter paper in Petri dishes at 23 °C. Those kernels that lacked a visible embryo were studied as a whole after removing the outer pericarp. If the embryo was visible, it was excised from the kernel after completion of imbibition and studied separately.
Germination of isolated embryos from fresh or imbibed kernels (50 on average) was carried out in Petri dishes on 0.7% agar in water. Scutellum and axis lengths were determined from electron micrographs. Water contents were analysed by weighing the samples before and after heating at 96 °C for 36–48 h.
EPR spin probe study
Membrane integrity was measured with an EPR spin probe technique, particularly suitable for small samples (Golovina and Tikhonov, 1994; Golovina et al., 1997). The excised embryos from imbibed kernels or whole imbibed kernels were incubated for 15–30 min in a 1 mM solution of the stable free radical, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPONE, Sigma, St Louis, MO, USA), containing 120 mM of the broadening agent K3Fe(CN)6. The sample was then loaded into a capillary (2 mm diameter) together with a small amount of the incubation solution. EPR spectra were recorded at room temperature with an X-band EPR spectrometer (Bruker, Rheinstetten, Germany, model 300E). Microwave power was 2 mW, and the modulation amplitude was 1 Gauss.
Principle of the EPR spin probe technique used to determine cellular membrane integrity
The spectrum of 1 mM TEMPONE in water is shown in Fig. 1A. This spectrum contains three equidistant narrow resonance lines accompanied by small satellites at both sides of each line resulting from the natural presence of 13C in TEMPONE molecules (ratio of satellite to main peak is 0.04; Marsh, 1981). The addition of K+-ferricyanide to a final concentration of 120 mM causes broadening to apparent invisibility (Fig. 1B) because of paramagnetic interactions (Eaton and Eaton, 1978). Placing a fresh 10 daa embryo into this broadened solution of TEMPONE causes the reappearance of the EPR signal (Fig. 1C), due to the differential permeability of the plasma membranes to TEMPONE molecules and ferricyanide ions. TEMPONE easily penetrates cells because of its amphiphilic nature and relatively small size. In contrast, the charged 
ions cannot penetrate through intact membranes (Keith and Snipes, 1974; Morse, 1977; Berg and Nesbitt, 1979). Thus, the spectrum presented in Fig. 1C has an intracellular origin, whereas the signal from TEMPONE located outside cells is still broadened by ferricyanide ions. In the case of membrane disruption, ferricyanide ions can penetrate cells and broaden the intracellular signal. Thus, the presence of cells with intact membranes in a sample can be determined by EPR in TEMPONE–ferricyanide solution. The sensitivity of the method allows the study of individual wheat embryos even if they are very small.
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Spectra subtraction
The EPR spectra of TEMPONE in cells containing oil bodies consist of two components originating from TEMPONE located in cytoplasm and in oil (see Results). Different splitting constants (the distance between lines) and different g-factors for hydrophobic and hydrophilic environments allow the resolution of the two components in the high-field region (right side) of the spectrum (Marsh, 1981, for references). Multi-component spectra must be decomposed to estimate the intensity and the rotational correlation times (
R) for each individual component properly. Decomposing was carried out by spectral titration, i.e. subtraction after adjusting for position and amplitude of the peaks (Berliner, 1976). Details of the decomposition of spectra are described in the Results section.
Calculation of rotational correlation times
The mobility of TEMPONE in cytoplasm depends on the viscosity of the micro-environment and can be quantified as the R (in seconds) that can be calculated from the cytoplasmic component of the EPR spectra. In the case of isotropic motion in the fast motional range the following equation was used (Knowles et al., 1976):
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R of a sample and R of water was used to show the relative increase in viscosity of the cytoplasm.
Intensity of EPR spectra
The intensity of the aqueous cytoplasmic and lipid components was calculated according to the following equation (Marsh, 1981):
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Low Temperature Scanning Electron Microscopy (LTSEM)
A LTSEM (JEOL, model 6300 F, Tokyo, Japan) was used to examine the material. The samples were mounted on stubs with conductive carbon cement (Leit-C, Neubauer Chemikalien, Münster, Germany). The stubs were placed on a specimen holder and subsequently frozen in liquid nitrogen (-196 °C). In the case of isolated embryos, excision and mounting were carried out in a box with vapour-saturated air. The frozen samples were transferred into a cryo-transfer unit (CT 1500 HF, Oxford Instruments, Oxon, UK). This unit consisted of a cryo-preparation chamber at high vacuum (10-6 Pa) dedicated to the LTSEM and a cryo-stage inside the microscope. The specimens were placed inside the cryo-chamber at -85 °C, kept there for 2 min to sublimate the contaminating water vapour. If required, the sample was cross-sectioned with a cold sharp knife. Finally the sample was sputter-coated with 3 nm platinum. The coated specimens were placed inside the LTSEM and observed at 1–5 kV. The temperature of the specimen inside the LTSEM was kept at -180 °C.
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Results |
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Normal kernel development on the mother plant
To appreciate the altered development of wheat kernels that were prematurely dried on the ear, normal development under greenhouse conditions is shown first. The changes in embryo length, dry weight, germination, and MC are shown in Fig. 2A
–D. For the staging of normal kernel development, the morphological staging of wheat caryopsis development was used (Rogers and Quatrano, 1983). Only from 10 daa onward, was it possible to excise fresh embryos from the surrounding tissues. The period until 10 daa is referred to as stage I, which is characterized by the occurrence of undifferentiated embryonic tissues. Between 10 and 15 daa, there was an increase in scutellum and axis lengths without considerable dry weight gain (stage II; Fig. 2A, B), during which the appearance of the embryo changed from transparent to opaque. At the end of this stage, between 13 and 15 daa, the fresh embryos acquired germinability (Fig. 2C), which requires the completion of at least a basic level of organ development. Indeed, at the end of stage II, scutellum, coleoptile, coleorhiza, primary root primordium, and leaf primordia have already been reported to be present (Rogers and Quatrano, 1983). Between 15 and 22 daa (stage III), the embryo dry weight rapidly increased from 0.2 mg to 1.1 mg on average, whereas the scutellum and axis lengths only slightly increased further (Fig. 2A, B). Desiccation tolerance, defined as the ability of excised embryos to germinate after fast drying, was acquired at 18 daa (Fig. 2C), approximately 4 d before the embryos reached the stage of mass maturity (maximum dry weight in Fig. 2B). The period between the attainment of maximal dry weight and beginning of maturation drying is considered as stage IV, lasting from 22–28 daa. Maturation drying (stage V) as seen in the kernel begun at approximately 28 daa (Fig. 2D), which is 10 d later than the acquisition of desiccation tolerance.
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0.05) when they diverge by 22% or more (
2-test). (D) Water content of the kernel and embryo during normal development (below 40% MC embryos could not be dissected without damaging them); the water content of kernels drying on the ear (of plants cut at 5 daa) is also indicated (open triangles). Embryo MCs were determined on 4–5 samples (approximately 30 embryos each), CV=1–5%; kernel MCs were determined individually (32 on average, CV ranging from 4–13%). The dashed line (in stage III) highlights the acquisition of desiccation tolerance during normal development. Stars indicate the scutellum length (A), dry weight (B) and germination (C) of embryos that were isolated from kernels that were dried on the ear of plants cut at 5 daa. Stages of development are according to Rogers and Quatrano (Rogers and Quatrano, 1983To obtain an insight into the physiological and structural changes during normal embryo development, an EPR spin probe technique and LTSEM were applied. Figure 1C
shows a representative EPR spectrum of TEMPONE in a fresh wheat embryo at 10 daa, which resembled the spectrum of TEMPONE in water (Fig. 1A). The intensity of the signal and the R value for TEMPONE in the cytoplasm were calculated (Table 1). The value of R was twice as high as that for the spin probe in aqueous solution. LTSEM micrographs of a fresh 10 daa embryo and its cryo-fractured cells are shown in Fig. 3A, B. The embryo axis was differentiated, but was considerably smaller than the scutellum. All the cells contained large vacuoles and clearly were undergoing cell division, which can be concluded from the image showing pairs of cells enveloped by a thick cell wall (Fig. 3B). This coincided with the increase in embryo length as shown in Fig. 2A. Protein and lipid bodies were scarce, which is consistent with the lack of dry weight accumulation (Fig. 2B). The lack of oil bodies can also be derived from the absence of a lipid component in the EPR spectrum from the 10 daa embryo (Fig. 1C; for the position of the lipid peak, see Fig. 4A).
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Figure 4A
shows a representative EPR spectrum from a fresh 14 daa embryo. At this stage of development, the increase in embryo length was almost completed (Fig. 2A), but desiccation tolerance was not acquired yet (Fig. 2C). The shape of the spectrum in Fig. 4A differed from that of the spectrum from the 10 daa embryo (Fig. 1C) in that two components were present, one originating from TEMPONE in aqueous cytoplasm and another originating from TEMPONE in lipid surroundings. These components are resolved at the right side (high-field region) of the spectrum (Golovina et al., 1997). The lipid peak overlaps the left satellite of the aqueous peak, for which no correction was made because the amplitude of the lipid peak was not influenced by the satellite. A typical EPR spectrum of TEMPONE in a 14 daa embryo that was fast-dried and rehydrated is shown in Fig. 4B. The spectrum mainly consisted of a lipid component, which can be derived from the distance between peaks (14.6 G versus 16.1 G for aqueous surroundings). The aqueous cytoplasmic component was considerably reduced as a result of the broadening by ferricyanide ions that had penetrated into the cytoplasm through disrupted plasma membranes. Ferricyanide ions do not broaden the signal of TEMPONE located in the lipid phase, because they cannot partition into this phase (Golovina et al., 1997, 1998). The plasma membrane disruption of the rapidly dried 14 daa embryo coincided with the loss of viability (Fig. 2C). Such dead embryos were characterized by the lack of turgidity of the cells and the absence of any internal structure (Fig. 5A, B). Images of fresh 14 daa embryos showed that the large vacuoles had disappeared (micrographs not shown).
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Information about the total cytoplasmic volume and viscosity can be obtained from the intensity and shape of the EPR spectrum of TEMPONE located in the cytoplasm. Because the cytoplasmic and lipid components are superimposed (Fig. 4A
), it is impossible to estimate these parameters directly. Therefore, the spectrum first had to be decomposed into its components. Assuming that the shape of the lipid component does not change with embryo development or drying, the lipid component can be used for subtraction to obtain the cytoplasmic component. For this purpose the spectrum of the rapidly dried 14 daa embryo (Fig. 4B) was used, which mainly consisted of the lipid component. By subtraction of the adjusted lipid component from the original spectrum (Fig. 4C, high-field region of the spectrum), the aqueous cytoplasmic component (Fig. 4D) was obtained. Because of the small intensity of the spectra to be subtracted, probable errors were minimized. The legitimacy of such subtractions is supported by the regular shape of the resulting aqueous cytoplasmic component (Fig. 4D).
The 5-fold increase in intensity of the cytoplasmic component from 10 to 14 daa (Table 1) indicates that the total cytoplasmic volume of the embryo increased about 5-fold. Table 1 and Fig. 4A further show that accumulation of oil already had begun at 14 daa, which coincides with the beginning of dry weight accumulation (Fig. 2B). The R in the cytoplasm of the 14 daa embryos was not significantly different from that of the 10 daa embryos (Table 1).
Figure 6A shows the EPR spectrum of TEMPONE in a fresh 18 daa embryo. After subtracting the adjusted lipid component (spectrum of Fig. 4B) from this spectrum, the aqueous cytoplasmic component was obtained (spectrum not shown) from which R was calculated. The R value from the fresh 18 daa embryo was significantly higher than that from the 14 daa embryo (Table 1). The EPR spectrum of TEMPONE in an 18 daa embryo that was fast-dried and rehydrated, contained a considerable cytoplasmic component, indicating that basically, membrane integrity was maintained (Fig. 6B). This correlates with the acquisition of desiccation tolerance (Fig. 2C). The R that was calculated from the aqueous cytoplasmic component (obtained by subtraction of the lipid component as outlined before) was as high as that before drying (Table 1). These data indicate that the acquisition of desiccation tolerance coincided with an increased cytoplasmic viscosity in the fully hydrated cells, possibly as the result of the accumulation of proteins and sugars (for references, see Bewley and Black, 1994; Dure, 1997). Representative images of a fresh 18 daa embryo and its cryo-fractured cells are shown in Fig. 3C, D. The embryo axis has approximately the same size as the scutellum. Protein and lipid bodies are abundant, and large vacuoles are not observed. Upon drying and rehydration of the 18 daa embryos, cells had a similar appearance as those of the non-dried, fresh control (micrograph not shown). The above-mentioned ultrastructural features accompanied the acquisition of desiccation tolerance.
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As a reference, the same procedures were followed for a fresh 28 daa embryo that can be considered as completely mature (Fig. 6C
). The intensity data in Table 1 indicate that there was no further increase in total cytoplasmic volume between 14 and 28 daa. This is also in agreement with the constant embryo length within this period of time (Fig. 2A). Meanwhile, the intensity of the lipid component increased 2.5-fold, which correlates with the dry weight accumulation (Fig. 2B). The R, calculated from the aqueous cytoplasmic component of spectrum C (Fig. 6), increased 1.5 times when compared with that in the fresh 14 daa embryo (Table 1). Electron microscopical inspection of the mature 28 daa embryos revealed the same features as found in the 18 daa embryos (micrographs not shown). Thus, growth parameters and germination tests are in good agreement with the EPR data, as well as with the LTSEM micrographs. In addition, EPR and LTSEM gave additional information about the changes in cytoplasmic viscosity, membrane integrity preservation, and ultrastructure associated with embryo maturation.
Premature drying of wheat kernels on the ear
Premature drying on the ear considerably changed the time-course of embryo development. Water contents of the kernels on cut plants passed through a similar decrease as those on intact plants, at least for the first 4–5 d after cutting (Fig. 2D). Thereafter, kernel MC content on cut plants declined more rapidly. Small excisable embryos could be found in kernels that were dried on the ears from plants cut as early as 5 daa, but not at an earlier stage. Such dwarf embryos (designated ear-dried 5 daa embryos) had a reduced scutellum length (0.90±0.12 mm) and embryo dry weight (0.144± 0.012 mg) when compared with normally developed, mature 22 daa embryos (2.08±0.16 mm and 1.055± 0.074 mg, respectively; Fig. 2A, B). Morphologically, they resembled a fresh 10 daa embryo, when all main organs are present (Fig. 3E, A). However, in contrast to the fresh 10 daa embryos, the ear-dried 5 daa embryos were germinable (Fig. 2C), although the seedlings had no lateral roots. Cryo-fractured cells of these dwarf embryos after rehydration had similar features as those of fresh desiccation-tolerant 18 daa embryos (Fig. 3F, D).
An EPR spectrum of TEMPONE in such a rehydrated ear-dried 5 daa dwarf embryo is shown in Fig. 6D. The intensity was relatively low when compared with that from a mature 28 daa embryo, but slightly higher than for the fresh 10 daa embryo (Table 1). The EPR spectrum from the dwarf embryo showed the presence of a lipid peak (see inset of Fig. 6D, for comparison with the amplitude and position of the satellite peak) which was absent from the EPR spectrum from the fresh 10 daa embryos (cf. Fig. 6D, 1C; Table 1). The R of the spin probe in the aqueous cytoplasm of the dwarf embryo was approximately 2-fold higher than that determined for the fresh 10 daa embryo (Table 1). Together, the oil accumulation, the increased cytoplasmic viscosity, the ultrastructural features, and the desiccation tolerance indicate that the ear-dried 5 daa dwarf embryo had passed through maturation during drying on the ear.
Acquisition of cellular desiccation tolerance in poorly developed embryos
In wheat kernels that were ear-dried earlier than 5 daa, embryos were never found by outside inspection. Such kernels never germinated. The same surprisingly sharp transition between 4 and 5 daa in the ability to develop dwarf embryos during ear-drying has been described for rye (Nutman, 1941) and barley (Harlan and Pope, 1922). Apparently, the initial stage of embryo development was so immature, i.e. a few cell divisions accomplished (Nutman, 1941; Batygina, 1987), that the minimally required development for germinability could not be completed during the period of ear-drying. Whether desiccation tolerance nevertheless could be acquired by such poorly developed embryos was studied using the EPR spin probe method and LTSEM.
Figure 7A and B show spectra of TEMPONE in two different ear-dried 4 daa kernels that did not contain an embryo that could be isolated. These spectra differed from the spectrum from the 5 daa embryo (Fig. 6D) in that they had a broad component superimposed onto the narrow ones. They also differed from one another in the ratio between the narrow and broad components. To be able to interpret these differences, the spectra had to be decomposed. For that purpose the shape of at least one of the components has to be known. Supposing that the shapes of the broad and narrow components were similar in both spectra and that these spectra only differed in the ratio between the components, spectrum (B) was subtracted from spectrum (A) in Fig. 7 after adjustment for amplitude and position of the broad component (Fig. 7C). The resulting difference spectrum (Fig. 7E) had a shape similar to the spectra obtained from the viable mature and dwarf embryos (Fig. 6C, D), containing only lipid and aqueous cytoplasmic components. From the presence of such a spectrum (Fig. 7E), it is concluded that viable cells with intact plasma membranes must be present in ear-dried 4 daa kernels without developed embryos.
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The broad component of the spectra can be obtained by subtracting spectrum (A) from spectrum (B) in Fig. 7
, after adjusting for amplitude and position of the narrow cytoplasmic components (Fig. 7D). The resulting spectrum (Fig. 7F) represents a broad signal that is always observed in spectra from dead tissues (Golovina et al., 1997). Such a type of spectrum can be the result of the presence of biopolymers that restrict the collisions of ferricyanide ions with TEMPONE molecules and reduce the broadening effect (Keith et al., 1977). This broad component can be subtracted from the original spectra (Fig. 7A, B) to obtain the spectrum from viable cells in the two ear-dried 4 daa kernels. Thus, the shape of the original spectra from the two ear-dried 4 daa kernels depends on the contribution of dead and viable tissues. Further subtraction of the lipid component (Fig. 4B) according to the method described above gave spectra of TEMPONE in the aqueous cytoplasm (Fig. 7G, H). The R values were even higher than those found for the other desiccation-tolerant embryos (Table 1).
To localize the viable cells in such an ear-dried 4 daa kernel, the embryonic end was cut from the distal end, and EPR spectra from both parts were recorded separately. Both spectra (Fig. 8A, B) contained narrow components indicating that viable cells were present in both parts of the kernel, albeit that the amplitude of the EPR signal was considerably higher at the embryonic end. Subtraction of the broad component (from Fig. 7F) following the procedure described above, gave spectra originating from viable cells (Fig. 8C, D). Because the distal end also contained viable cells, it is concluded that beside embryonic cells, some endosperm cells also can acquire desiccation tolerance.
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To compare the cytoplasmic viscosity in these desiccation-tolerant endosperm cells with that in the embryonic cells, the aqueous cytoplasmic component was obtained as outlined before (Fig. 8E
, F) and R values were calculated. From the similar R values (indicated in Fig. 8E, F), the presence of the lipid component (oil bodies), and the ability to maintain membrane integrity upon dehydration, it is concluded that during drying on the ear, some endosperm cells could pass through the same cytoplasmic changes as do embryonic cells.
Figure 9A to D show LTSEM images of cryo-fractured, ear-dried wheat kernels with a poorly developed embryo (4 daa) that was invisible by external inspection. The kernels were rehydrated on filter paper before plunging them in liquid N2 and loading into the LTSEM. The overview of the cryo-fractured sample shows a non-differentiated axis and the crease region of the endosperm (Fig. 9A). The turgid surface of the cells from the poorly developed embryo indicates that the plasma membranes are intact and that the cells survived dehydration-rehydration (Fig. 9A–C). The cryo-fracture images further show that these cells are filled with protein and lipid bodies and do not contain large vacuoles. Obviously, these cells contribute to the narrow components in the EPR spectra from the embryonic end of the kernel (Fig. 8C).
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Other viable cells were localized at the periphery of the endosperm (Fig. 9D
). They are large, also filled with protein and lipid bodies, and do not contain large vacuoles. Obviously, these cells are responsible for the narrow cytoplasmic component of the EPR spectrum in the distal end and partly in the embryonic end of the kernel (Fig. 8C, D). They are 2–3 times larger (50–60 µm) than the embryonic cells (15–20 µm) and are located in 2–3 layers on the periphery of the endosperm. These peripheral cells are different from other endosperm cells that have the typical appearance of dead cells of the starchy endosperm (Fig. 9D). Thus, LTSEM micrographs support the data obtained with the EPR investigation indicating that even in ear-dried 4 daa kernels desiccation tolerance was acquired in the poorly developed embryos and peripheral (cambial) endosperm cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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During seed development, cell division and histo-differentiation are followed by maturation including the acquisition of desiccation tolerance (Walbot, 1978
; Kermode, 1990; Harada, 1997). The duration of every stage of development depends on species, variety and environmental conditions. During the normal wheat embryo development described in this work, the usual sequence of events was observed as previously established for wheat (Rogers and Quatrano, 1983; Symons et al., 1983; Rasyad et al., 1990; Black et al., 1996; Sanhewe et al., 1996). Desiccation tolerance was acquired approximately 3 d after the onset of the maturation stage and approximately 4 d before mass maturity, but considerably (10 d) before the beginning of maturation drying (Fig. 2A–D).
The transition from the desiccation-sensitive to the desiccation-tolerant stage is accompanied by structural and physical changes in the cells. Large vacuoles disappeared and protein and lipid bodies were formed (Fig. 3B, D). Indeed, the disappearance of the vacuoles as reported here and elsewhere (Farrant et al., 1997), is considered as one of the mechanisms of desiccation tolerance (Bewley, 1995; Pammenter and Berjak, 1999). Thus, mechanical damage associated with excessive changes in cell volume is avoided. Using an EPR spin probe technique and LTSEM, it is shown that maintenance of membrane integrity upon drying/rehydration and increased cytoplasmic viscosity correlate with desiccation tolerance (Table 1; Figs 3–6). Compositional changes in the cytoplasm, such as the accumulation of sugars or Lea-proteins (Blackman et al., 1992), may underlie the increase in viscosity.
These data on slow premature drying of wheat kernels on the ears from plants cut at 5 daa show that development continued for some time resulting in the formation of viable dwarf embryos, as shown before with rye and barley kernels (Nutman, 1941; Harlan and Pope, 1926). Morphologically, the dwarf embryo was slightly advanced when compared with the 10 daa, normally developed embryo (Fig. 3A, E), but with almost similar size and weight (Fig. 2A, B). This would mean that cell division and histo-differentiation might have continued for at least 5 d after excision, which is in agreement with the first noticeable water loss from the kernels after 4–5 d (Fig. 2D). At the cellular level, the dwarf embryo considerably differed from the fresh desiccation-sensitive 10 daa embryo. These 5 daa, ear-dried dwarf embryos are characterized by the absence of large vacuoles, the presence of protein and oil bodies, dense cytoplasm, increased cytoplasmic viscosity, and the retention of membrane integrity (Figs 3F, 6D; Table 1). The morphological development and cellular desiccation tolerance of the dwarf embryos permitted germination (Fig. 2C). The fact that also for the dwarf embryos the LTSEM and EPR results were in agreement with desiccation tolerance as derived from germination ability, allows these methods to be used to assess cellular desiccation tolerance in those cases in which kernels had insufficient morphological development for germination.
The absence of germination ability upon very early premature drying has previously led to the conclusion that the developing embryos did not survive dehydration (Nutman, 1941; Adams and Rinne, 1981; Dasgupta et al., 1982; Kermode and Bewley, 1985). However, the inability to germinate does not unambiguously prove that embryonic cells are desiccation sensitive. Some evidence that poorly developed embryos may have survived dehydration came from studies on maize (Sheridan and Clark, 1993) and Arabidopsis thaliana (Devic et al., 1996) embryo-defective mutant seeds that are blocked at early stages of embryogenesis. The fact that such mutant embryos did not become necrotic following desiccation, together with the fact that they expressed genes associated with maturation (see Harada, 1997, for references), suggests that these poorly-developed embryos passed through maturation and acquired desiccation tolerance. Obviously, studying the acquisition of desiccation tolerance at the cellular level in wild-type seeds can give an answer to the question whether or not histo-differentiation is necessary for the acquisition of desiccation tolerance.
Evidence of the independence of the maturation programme from morphogenesis comes from our EPR and LTSEM data on wheat kernels that were dried so prematurely (4 daa ear-dried) that on first sight no embryos developed. However, the kernels contained poorly developed, non-germinable embryos that had acquired cellular desiccation tolerance (Figs 7, 9; Table 1). The turgidity of the rehydrated cells as a sign of membrane integrity (Fig. 9A–C) provides additional evidence of desiccation tolerance at the cellular level.
In addition to the embryonic cells, there were some endosperm cells in the 4 daa ear-dried kernels that survived dehydration (Figs 8D, F, 9D). These cells are located at the periphery of the endosperm. They have a meristematic function in producing starchy cells during endosperm growth. These starch-accumulating cells, did not have the competence to acquire desiccation tolerance (Fig. 9D). Later in development, the peripheral multi-layer of meristematic (cambial) cells will transform into one layer of aleurone cells (Evers, 1970; Batygina, 1987) that acquire desiccation tolerance. Apparently, before their differentiation into aleurone layer cells, the meristematic (cambial) endosperm cells also have the propensity to acquire desiccation tolerance when they are prematurely dehydrated.
In the present study it is demonstrated with very prematurely dried wheat kernels that cellular maturation processes in seeds, including the acquisition of desiccation tolerance, can be initiated considerably before the completion of cell division and histo-differentiation. It has been shown that a slight decrease of the MC, i.e. 15% on a dry-matter basis, can induce desiccation tolerance in developing wheat embryos (Black et al., 1999). Because morphogenesis and maturation cannot take place simultaneously, cell division has to be arrested before the maturation programme is initiated (Harada, 1997). It is envisaged that the slight water loss during slow drying on the ear, 4–5 d after cutting of the plant (Fig. 2D), can accomplish this shift in cell development. Indeed, DNA replication has been reported to be sensitive to water loss below the relatively high water content of approximately 75% (fresh weight basis) (Brunori, 1967). Because metabolic activity is less sensitive to water loss (Vertucci and Farrant, 1995; Leprince and Hoekstra, 1998), there is time during the slow drying to initiate a maturation programme that may continue until a critical MC is reached below which even this programme cannot proceed.
The present data indicate that meristematic cells of the poorly developed embryo and the meristematic (cambial) layer of endosperm have the competence to acquire desiccation tolerance upon the proper signalling. More evidence that non-differentiated cells can acquire desiccation tolerance comes from the observation that after a treatment with abscisic acid, callus from the resurrection plant, Craterostigma plantagineum, becomes desiccation tolerant (Bartels et al., 1990), which led to the suggestion that the vegetative parts of this resurrection plant can be considered as ‘eternal seed’. For orthodox seeds, it is suggested that the competence for the acquisition of desiccation tolerance is present immediately upon fertilization and lost with the transition to vegetative growth.
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This work was supported in part by grant No. 047.01.006.96 from the Netherlands Organization for Scientific Research and NATO collaborative linkage grant No. LST.CLG 975082.
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4 To whom correspondence should be addressed. Fax: +31 317 484740. E-mail: folkert.hoekstra{at}pph.dpw.wau.NI
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