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Plant Physiol. (1999) 120: 153-164 Changed Properties of the Cytoplasmic Matrix Associated with Desiccation Tolerance of Dried Carrot Somatic Embryos. An in Situ Fourier Transform Infrared Spectroscopic Study1
Laboratory of Plant Physiology, Wageningen Agricultural University, Arboretumlaan 4, NL-6703 BD Wageningen, The Netherlands (W.F.W., M.A., F.A.H.); and Incotec, Westeinde 107, NL-1601 BL Enkhuizen, The Netherlands (F.A.A.T.)
Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be "less tight" in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesis abundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos.
Desiccation tolerance is the capacity of an organism or tissue to
regain vital metabolism after almost complete dehydration. Seeds,
pollens, resurrection plants, mosses and ferns, nematodes, tardigrades,
yeasts, fungi spores, and bacteria have this capacity (for review, see
Crowe et al., 1992 Carrot (Daucus carota) somatic embryos can be rendered
tolerant to severe desiccation by a proper combination of treatments (Tetteroo et al., 1994 Large amounts of soluble carbohydrates have been suggested to be
involved in the acquisition of desiccation tolerance (Crowe et al.,
1984 Sugars may act as protectants of proteins and membranes in dehydrating
seeds (Crowe et al., 1987 Recently, we applied in situ FTIR to assess the heat stability of
proteins and the glassy cytoplasmic matrix in anhydrobiotic organisms
such as pollen (Wolkers and Hoekstra, 1997 We used in situ FTIR to study the heat stability of proteins and
properties of the glassy matrix in slowly dried, desiccation-tolerant, and rapidly dried, desiccation-sensitive carrot somatic embryos. To
explain the desiccation tolerance of the slowly dried somatic embryos
by the considerably increased umbelliferose content, we analyzed
the protecting properties of purified umbelliferose in model
systems, which were compared with those of Suc. The roles of
umbelliferose and de novo synthesized proteins in the acquisition of
desiccation tolerance are discussed.
Production, Drying, and Germination of Somatic Embryos
Extraction and Purification of Umbelliferose Dry somatic embryos (10 g) were boiled in 200 mL of 80% methanol for 1 h. The embryos were then filtered and washed with 50 mL of 80% methanol, and the filtrates were combined. The methanol was removed from the filtrate by vacuum evaporation, and we defatted the remaining aqueous suspension by passing it through a C18 reversed-phase column (Waters). We then purified the suspension by passing it through a column of Polyclar AT (insoluble PVP, BDH Chemicals Ltd., Poole, UK). After the volume of the suspension was reduced, it was layered onto Sephadex QAE-A-25-formate and Sephadex SP-C-25-H+ columns (Pharmacia) and eluted with ultrapure water (Milli-Q system, Millipore) according to the method of Redgwell (1980) . The eluate containing the sugar fraction was freeze-dried to
reduce volume. If required, the Sephadex column-purification procedure was repeated. Umbelliferose was separated from the other sugars by
preparative HPLC on a Shodex OH pak Q-2002 column (20 × 500 mm;
Waters) by using ultrapure water at 55°C as the eluant at 3 mL
min 1 and a refractive index detector (model SP 8430, Spectra Physics, San Jose, CA). Using HPLC (analytical PA-1 column,
9 × 250 mm, Dionex, Sunnyvale, CA; see also "Carbohydrate
Analysis") we further purified the umbelliferose. The NaOH in the
eluant recovered from the column was removed using an anion
self-generating suppressor (4 mm, Dionex) and 50 mM
H2SO4 as the regenerant, with a flow rate of 5 mL min1. The purified umbelliferose solution
was then lyophilized to be used for the FTIR measurements and to
determine the response factor for the quantitative analysis of
umbelliferose by HPLC. The umbelliferose preparation purified according
to the above procedure was characterized by one single peak in the HPLC
chromatogram. Alternatively, extractions were made starting from
50 g of seed that was ground in 80% methanol in a mortar with a
small amount of sand.
Carbohydrate Analysis Per lot of lyophilized somatic embryos, approximately 10 mg was mixed with 1 mL of 80% methanol containing 1 mg of raffinose as the internal standard. The samples were kept at 76°C in a water bath for 15 min to extract the soluble carbohydrates and to inactivate enzymes. Subsequently, the methanol was evaporated in a Speedvac (Savant Instruments, Farmingdale, NY). The samples were then suspended in 1 mL of ultrapure water. After centrifugation in an Eppendorf centrifuge, the supernatants were diluted 50 times for HPLC analysis.IR Spectroscopy FTIR spectra were recorded on an IR spectrometer (model 1725, Perkin-Elmer) equipped with a liquid N2-cooled mercury/cadmium/telluride detector and a microscope (Perkin-Elmer) as described previously (Wolkers and Hoekstra, 1995).
Preparation of Liposomes for FTIR Analysis and Leakage Studies Egg PC in CHCl3 (Fluka) was used without further purification. After the CHCl3 was removed in a vacuum overnight, the dry egg PC was rehydrated in water at a concentration of 10 mg mL1. When required,
carbohydrates were added externally to the egg PC suspension to give a
mass ratio of 5:1 (sugar:egg PC). Subsequently, we produced unilamellar
vesicles by passing the suspension 35 times through one 100-nm-pore
size polycarbonate filter (Nuclepore, Pleasanton, CA) as described
previously (Van Bilsen et al., 1994). For IR spectroscopy, 5-µL
samples were dried directly on circular CaF2
windows (13 × 2 mm) for at least 3 h in a stream of dry air at 23°C (<3% RH). Before the samples were removed from the dry-air box, another window was placed on top of the sample window, with a
rubber ring in between. Hydrated liposome samples were concentrated by
ultracentrifugation, and the pellet was used for FTIR analysis.
RNA Extraction and Northern Hybridization For the isolation of RNA, 30 to 50 mg of embryo material was taken at 24-h intervals during slow drying, starting at 0 h, and then rapidly dried for 4 h in a flow cabinet. Subsequently, the embryos were ground with a mortar and pestle in the presence of liquid N2. Lysis of the material was done in 50 mM Tris-HCl buffer, containing 0.5 M NaCl, 50 mM EDTA, and 10 mM -mercaptoethanol, pH 8.5. Subsequently, the mixture was homogenized in a mixture of 6%
2-butanol, 1% triisopropylnaphtalene disulfonate, 2%
para-aminosalicylate, and 4% SDS and extracted with phenol:chloroform
(1:1, v/v). RNA was precipitated overnight with 2 M LiCl,
and poly(A+) RNA was obtained by affinity
chromatography on oligo(dT)-cellulose. Poly(A+)
RNA was electrophoresed on a glyoxal/DMSO gel and blotted onto a nylon
membrane (Gene Screen Plus, DuPont) as described by Sambrook et al.
(1989). Hybridization was done using a random-primed DNA-labeling kit
(Boehringer Mannheim). The dehydrin clone B18 (Close et al., 1989) was
used as the probe.
In Situ Protein Secondary Structures as Influenced by Drying Rate Somatic embryos grown in a Suc- and ABA-containing medium were subjected to slow and fast drying. Slowly dried embryos usually germinated for more than 90%, whereas the germination of the rapidly dried embryos varied between 0% and 30%. Figure 1A depicts the in situ IR spectra of slowly and rapidly dried somatic embryos in the 1800 to 1500 cm1 region. The band around 1740 cm1 in this region is attributable to ester
bonds arising from lipids. The differences in peak height around 1740 cm1 were the result of uncontrolled losses of
neutral lipid during the sandwiching of the sample between the diamond
windows and were not taken into consideration any further. The amide-I
band around 1650 cm1 and the amide-II band
around 1550 cm1 arise from the protein backbone
(Wolkers and Hoekstra, 1995). For protein structural studies we have
focussed on the amide-I band. Slight but consistent differences can be
observed between the amide-I band profiles of slowly and rapidly dried
somatic embryos. The slowly dried embryos had a relatively stronger
absorption around 1654 cm1 than the rapidly
dried embryos. Sometimes, a clear band around 1632 cm1 was observed in spectra of rapidly dried
somatic embryos, which may be indicative of intermolecular protein
clusters (denaturation) or protein breakdown.
Heat Stability of Endogenous Proteins When dry, intact tissues are heated and monitored with respect to protein secondary structure, information can be obtained concerning the intrinsic heat stability of the proteins in their native environment (Wolkers and Hoekstra, 1997; Wolkers et al., 1998a). In the slowly and
rapidly dried somatic embryos, the band at approximately 1637 cm1 shifted with temperature to approximately
1630 cm1, which is characteristic of
intermolecular extended -sheet structures (Fig.
2). This can be interpreted as protein
denaturation and is irreversible, i.e. after cooling the bands did not
return to their original positions. Figure 2 also shows that the peak
height at 1630 cm1 in spectra of the
heat-denatured, rapidly dried embryos was more pronounced than in those
of the slowly dried embryos. This indicates that the extent of protein
denaturation is greater in the rapidly dried somatic embryos. The
 -helical band at approximately 1657 cm1
hardly shifted in position with temperature for both drying treatments. The heat-induced protein Td was derived
from a plot of the position of the turn/-sheet band (1637 cm1) versus the temperature (Fig.
3). In the slowly and rapidly dried embryos, the band position sharply fell to lower wave numbers above
90°C, indicating that denaturation had begun in both types of
embryos. Td values, which were derived from
Figure 3 with the help of first-derivative analysis of the curve fitted
through the data, were 109°C and 107°C for slowly and rapidly dried
embryos, respectively.
Properties of the Dry Cytoplasmic Glassy Matrix FTIR was used to study the glassy matrix in the slowly and rapidly dried somatic embryos. For this purpose, the OH (at
approximately 3330 cm1), arising mainly from
the sugar OH groups, was monitored as a function of temperature
(Wolkers et al., 1998c). Two linear regression lines could be drawn in
the OH-versus-temperature plot of the slowly dried embryos
with an intersection point at 48°C (Fig. 4). The temperature at the intersection
point can be considered as Tg. This
indicates that the slowly dried embryos are in a glassy state at room
temperature. The slopes of the regression lines, the WTC values, were
0.14 and 0.38 cm1/°C below and above
Tg, respectively, and are indicative of the average strength of the hydrogen-bonding interactions. In the rapidly
dried embryos no clear break in the OH-versus-temperature plot could be observed, which indicates that there is not one defined
Tg. Moreover, the WTC of 0.30 cm1/°C in the rapidly dried embryos below 40°C
was much higher than that of the slowly dried ones. The higher WTC of
the rapidly dried embryos suggests a less tight hydrogen-bonding
network, which is associated with a more loosely packed glassy
structure in these embryos.
Effect of Drying Rate on the Composition of Soluble Carbohydrates It was reported previously that slow drying leads to the accumulation of the trisaccharide umbelliferose in carrot somatic embryos (Tetteroo et al., 1994, 1995). This accumulation does not occur
when the embryos are subjected to fast drying. Because umbelliferose,
apart from Suc, may make up a considerable portion of the total dry
weight, an important role was attributed to this trisaccharide in the
stabilization of the dry cytoplasm. During protocol development for
optimizing desiccation tolerance of the somatic embryos, a large number
of different treatments were given. The variables in these experiments
were the genotypes (cvs RS 1 and Trophy) and the concentration
of added ABA and Suc in the maturation medium. Whereas omission of ABA
from the maturation medium and fast drying led to reduced viability,
the ABA and Suc concentrations used resulted in optimal survival of the
somatic embryos. Figure 5 shows a plot of
the umbelliferose against the Suc contents in each individual lot of
slowly dried embryos that had germination percentages of 95% or more.
The contents of Suc and umbelliferose in a few rapidly dried embryo
lots of poor viability (10%-28%) are also shown. It is clear that
slow drying leads to increased contents of umbelliferose. However, the
amounts observed were variable, without apparent effect on desiccation
tolerance. The linear regression coefficients of the lines that can be
drawn through the data points for both cultivars (r =  0.89 and
0.78 for cvs RS 1 and Trophy, respectively) suggest that
umbelliferose is produced at the expense of Suc. The apparent
interchangeability between these sugars and the generally excellent
survival after drying suggest further that Suc and umbelliferose may be
equally important in relation to desiccation tolerance.
Comparison of Stabilizing Properties of Umbelliferose and Suc In an attempt to ascribe the changes in physical stability as observed by in situ FTIR to the elevated amounts of umbelliferose in the slowly dried embryos, several properties of umbelliferose were tested in model systems and compared with those of Suc.Glass-Forming Properties The glass-forming properties of pure umbelliferose and Suc were monitored using FTIR, as was done for the intact somatic embryos. Figure 6 shows theOH-versus-temperature plots of umbelliferose and Suc, from
which the Tg values for dry umbelliferose
and Suc glasses were determined at 66°C and 60°C, respectively.
Before the measurements, the samples were heated to 80°C for several minutes, in which all water detectable in the IR spectra was removed. The WTC values in the glassy state were 0.20 cm1/°C for umbelliferose and Suc.
Protein Protection One hypothesized role of sugars in desiccation tolerance is interaction during drying with proteins, which would prevent dehydration-induced conformational changes (Carpenter and Crowe, 1989;
Wolkers et al., 1998d). To study whether umbelliferose is effective in
this respect, we selected poly-L-Lys, a synthetic
polypeptide that undergoes structural changes with freeze-drying
(Prestrelski et al., 1993) and air-drying (Wolkers et al., 1998d). As
in Figure 6, the samples were heated to 80°C for several minutes
before the measurements. When poly-L-Lys was air-dried in
the presence of umbelliferose, a broad band at 1653 cm1 dominated, representing a random coil
structure (Fig. 7). A similar spectrum
can be observed when poly-L-Lys is dried in the presence of
Suc (Wolkers et al., 1998d). Without carbohydrate, drying led to
absorption bands at 1625 and 1695 cm1, which
are indicative of extended -sheet structure. In the hydrated state
poly-L-Lys exists entirely in the random coil conformation (Tiffany and Krimm, 1969; Jackson et al., 1989; Wolkers et al., 1998d).
The above results show that umbelliferose, similar to Suc, can prevent
dehydration-induced conformational transitions of
poly-L-Lys.
Interactive Effect with Protein on Glassy Behavior Sugars and proteins may form a tight hydrogen-bonding network when dried together, which leads to increased molecular stability and elevated Tgs. We studied the glassy behavior of dried umbelliferose/poly-L-Lys mixtures (preheated) on the basis of the temperature-dependent shifts in the positions of the OH-stretching band (Fig. 8). Poly-L-Lys is particularly suitable in this respect, because it lacks OH groups, thus, only sugar OH groups were studied. With increasing amounts of the polypeptide in the sugar polypeptide mixture, Tg increased. The WTCg decreased from 0.20 to 0.05 cm1/°C in the range from 0 to 1 mg
poly-L-Lys/mg umbelliferose, respectively (Fig.
9). The decrease in
WTCg on addition of the polypeptide is
interpreted to mean that poly-L-Lys directly
interacts through hydrogen bonding with umbelliferose to form a
tightly packed molecular network. Suc acts similarly in this respect.
Protection of Liposomes Sugars in anhydrobiotic organisms may also play a role in the protection of membranes. By interacting with the polar headgroups of phospholipids, sugars were found to depress Tm of model membranes (Crowe et al., 1992,
1996). Using FTIR we measured the position of the absorption band
attributed to the CH2 symmetric stretching vibration of the acyl chains in egg PC liposomes. During the transition from the gel to the liquid crystalline phase, there is a wave number
shift from 2851 to 2854 cm1 (Hoekstra et al.,
1989). Figure 10 shows that
umbelliferose can prevent the dehydration-induced increase of
Tm in egg PC liposomes. Whereas hydrated
liposomes have a Tm at approximately
8°C and the air-dried liposomes at 32°C, the
Tm of liposomes dried in the presence of
umbelliferose was 35°C, far below that of the hydrated control. Suc
had an identical effect on the dry liposomes. Thus, the lipid bilayer
remains in the liquid crystalline phase during dehydration at 20°C,
which is one of the prerequisites for the protection of liposomes in
the dry state (Crowe et al., 1994, 1996, 1997b). Also, a slight shift
between 10°C and 50°C could be observed for both sugars, which
points to a slightly inhomogeneous interaction with the headgroups. The
samples were not preheated, because this would lead to fusion of the
liposomes (Crowe et al., 1997b).
Expression of a Dehydrin Transcript (B18) during Drying
Carrot somatic embryos acquire the capacity to become desiccation
tolerant by the addition of ABA to the maturation medium, but the
actual tolerance requires slow drying for several days (Tetteroo et
al., 1995 Protein Secondary Structure
Heat Stability of Endogenous Proteins During drying, the cytoplasm of the embryos transforms into a glassy matrix, which is thought to immobilize macromolecular and cellular structures, thus providing stability (Williams and Leopold, 1989; Leopold et al., 1994). Using FTIR we were able to assess in situ
the stability of endogenous proteins embedded in this dry glassy
matrix. No differences in protein denaturation temperatures between
rapidly and slowly dried embryos were found. However, the extent of
protein denaturation was greater in the rapidly dried embryos, as was
deduced from the greater relative proportion of irreversible protein
aggregates (Fig. 2). Apparently, proteins are immobilized to a lesser
extent in the rapidly dried embryos, which permits more heat-induced
protein-protein interactions (denaturation) in the dry cytoplasmic
matrix of these embryos.
Properties of the Viscous-Solid Matrix To link the differences in heat denaturation behavior between the slowly and rapidly dried embryos to possible differences in glassy behavior, heat-induced shifts in the OH stretch were investigated. We found that slowly dried somatic embryos were in a glassy state at room temperature (Tg = 48°C) and that no clearly defined Tg could be observed for the rapidly dried embryos. Furthermore, the WTC values below 40°C were considerably higher for the rapidly dried specimens than for the slowly dried ones. This means that the average strength of intermolecular hydrogen bonding, or the molecular packing density, is greater in the slowly dried embryos than in the rapidly dried embryos. The reduced molecular packing density may account for the reduced protein stability in the rapidly dried somatic embryos. High WTC values and less protein stability were also found in desiccation-sensitive, maturation-defective mutant seeds of Arabidopsis (Wolkers et al., 1998a).
Role of Umbelliferose in Slowly Dried Carrot Somatic Embryos Whereas Suc is the major soluble carbohydrate after fast drying (up to 20% of the dry weight), after slow drying, the trisaccharide umbelliferose accumulates at the expense of Suc up to 15% of the dry weight. During fast drying, time is lacking for the synthesis of umbelliferose. For seeds in general, the accumulation of oligosaccharides has been linked with a better long-term survival in the dry state (Horbowicz and Obendorf, 1994; Horbowicz et al., 1995).
Also, in the present work elevated umbelliferose contents evoked by slow drying correlated well with improved desiccation tolerance. Particularly, the ratio of Suc to oligosaccharides is considered important in this respect rather than Suc and the oligosaccharides in
their absolute amounts (Bernal Lugo and Leopold, 1995; Horbowicz et
al., 1995). It was suggested that even small amounts of
oligosaccharides prevent the crystallization of Suc (Caffrey et al.,
1988), thus improving survival in the dry state. However, in our work
the rapidly dried, desiccation-sensitive embryos still contained
umbelliferose for approximately 3% of the dry weight, which may have
prevented Suc crystallization.
Stabilizing Properties of Umbelliferose Compared with Suc Both umbelliferose and Suc form a glassy state upon air-drying. The Tg of umbelliferose was slightly higher than that of Suc, 66°C compared with 60°C, respectively. Both sugars are equally effective in depressing the Tm of dry egg PC liposomes. Thus, the liposomes remain in the liquid crystalline phase during dehydration at ambient temperatures; for protection of liposomes in the dry state, this is one of the prerequisites (Crowe et al., 1994, 1996). Another
prerequisite is prevention of liposome fusion, which depends on the
presence of good glass-forming compounds (Crowe et al., 1997b). As
discussed above, umbelliferose and Suc are examples of good glass
formers. It is therefore no surprise that both sugars are able to
retain CF inside liposomes after dehydration in their presence.
Likewise, raffinose and stachyose have this ability but monosaccharides
do not (Crowe et al., 1997b).
Possible Role of LEA Proteins versus Umbelliferose The rapidly dried somatic embryos are different from the slowly dried embryos in that they lack dehydration-induced protein synthesis and further accumulation of umbelliferose. Such proteins may have an important impact on the glassy matrix of the dry embryos. We previously showed that LEA proteins purified from pollen increased the Tg and the molecular packing density of a Suc glass (W.F. Wolkers, unpublished data). Based on the similar physical properties of Suc and umbelliferose, we suggest that the lesser physical stability of the rapidly dried embryos is most likely due to a lack of dehydration-induced proteins rather than to a lack of accumulated umbelliferose.
2 Present address: Section of Molecular and Cellular Biology, University of California, Davis, CA 95616. Received September 8, 1998;
accepted January 15, 1999.
We thank Dr. Renske van der Veen and Sander van Hal for their help with the northern hybridization experiments. We thank Dr. Henk A. Schols (Food Science Department, Wageningen Agricultural University, The Netherlands) and Dr. Steef M. De Bruijn (Laboratory of Plant Physiology, Wageningen Agricultural University, The Netherlands) for their help with the Dionex experiments.
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Hoekstra FA
(1998b)
Fourier transform infrared microspectroscopy detects changes in protein secondary structure associated with desiccation tolerance in developing maize embryos.
Plant Physiol
116:
1169-1177
Wolkers WF, Hoekstra FA (1995) Aging of dry desiccation-tolerant pollen does not affect protein secondary structure. Plant Physiol 109: 907-915 [Abstract] Wolkers WF, Hoekstra FA (1997) Heat stability of proteins in desiccation tolerant cattail pollen (Typha latifolia): a Fourier transform infrared spectroscopic study. Comp Biochem Physiol 117A: 349-355 [CrossRef] Wolkers WF, Oldenhof H, Alberda M, Hoekstra FA (1998c) A Fourier transform infrared microspectroscopy study of sugar glasses: application to anhydrobiotic higher plant cells. Biochim Biophys Acta 1379: 83-96 [Medline] Wolkers WF, Van Kilsdonk M, Hoekstra FA (1998d) Dehydration-induced conformational changes of poly-L-lysine as influenced by drying rate and carbohydrates. Biochim Biophys Acta 1425: 127-136 [Medline]
Copyright Clearance Center: 0032-0889/99/120//12
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 J. Yang and H. E. Yen Early Salt Stress Effects on the Changes in Chemical Composition in Leaves of Ice Plant and Arabidopsis. A Fourier Transform Infrared Spectroscopy Study Plant Physiology, October 1, 2002; 130(2): 1032 - 1042. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
O stretching vibration band (proteins) around 1635 cm
95%), slowly
dried carrot somatic embryos of the cvs RS 1 and Trophy. The
treatment variables were Suc and ABA concentrations in the maturation
medium. The filled symbols represent the Suc and umbelliferose content
after fast drying. DW, Dry weight.
