JXB Advance Access published online on November 17, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm250
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Changes in water status and water distribution in maturing lupin seeds studied by MR imaging and NMR spectroscopy
gorzata Garnczarska1,*
1Department of Plant Physiology, Adam Mickiewicz University, Umultowska 89, 61-614 Pozna
, Poland
2Department of Macromolecular Physics, Adam Mickiewicz University, Umultowska 85, 61-614 Pozna, Poland
* To whom correspondence should be addressed. E-mail: garnczar{at}main.amu.edu.pl
Received 11 May 2007; Revised 30 July 2007 Accepted 17 September 2007
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Abstract |
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The changes in water distribution in maturing lupin (Lupinus luteus L.) seeds were visualized with magnetic resonance imaging (MRI). MRI data showed local inhomogeneities of water distribution inside the seed. At the late seed-filling stage the most intense signal was detected in the seed coat and the outer parts of cotyledons in the hilum area, but during maturation drying the decline in MR image intensity was faster in the outer part of the seed than in the central part. The changes in water status were characterized by NMR spectroscopy. Analyses of T2 relaxation times revealed a three-component water proton system in maturing lupin seeds. Three populations of protons found during seed maturation, each with a different magnetic environment causing a different relaxation rate, were correlated with three fractions of water (structural, intracellular, and extracellular) that were observed during seed germination. This study provides evidence that lupin seeds have similar states of the different water components with regard to seed moisture content at two distinct physiological stages, seed maturation and germination. The unique feature of maturing lupin seeds is the presence of the high 1H-NMR signal in areas corresponding to the vascular bundles. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of vascular tissue. An anti-dehydrin antibody detected three polypeptides in lupin embryos with molecular masses of 73, 43 and 28 kDa, respectively. The temporal pattern of dehydrin protein accumulation correlates well with seed desiccation.
Key words: 1H-NMR, dehydrins, lupin, Lupinus luteus, MRI, seed maturation, T2 relaxation, water content
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Introduction |
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The development of orthodox seeds is terminated by the loss of water, which results in a gradual reduction in metabolism, and the embryo passes into a metabolically inactive or quiescent state (Kermode and Finch-Savage, 2002). This terminal phase of seed development, called maturation drying, is also known to ensure the switch from a developmental mode to a germinative mode (Kermode and Bewley, 1985). Although the occurrence and extent of this drying varies, in excess of 80% of cellular water is lost. As water is removed from the cells, the physical and physiological properties of the cells change. Removing water from the cells pushes the cellular constituents together and a consequence of this molecular aggregation is an increased ordering of molecular structures. Drying-induced compaction of molecules requires greater packing efficiency, resulting in the localized enrichment of similar molecules in a process known as demixing (Walters et al., 2002). Most seeds lose water as reserves are deposited primarily within storing tissues thus displacing water from the cells and then during maturation drying when fresh weight loss is accompanied by a rapid decline in seed water content. To date, few reports on the mechanism and route of water loss from seeds have been published. Some authors suggest the existence of a passive mechanism whereby water is lost primarily by evaporation from the surface of the seed (Nechiporenko and Rybakova, 1983; Lee and Atkey, 1984; Goncharova et al., 1985). As also reported, water may move from the seed to the parent plant by a metabolically active process (Meredith and Jenkins, 1975). Greenwood and Bewley (1982) showed that, in castor bean, desiccation is initiated by the severing of the vascular supply to the seed (funiculus detachment) and senescence of the capsule. According to Kermode and Finch-Savage (2002) this finding suggests that the relocation of water from the seed to the parent plant is not the means by which the water loss occurs.
The loss of water is either coincident with or subsequent to the acquisition of desiccation tolerance by the embryo. Desiccation tolerance corresponds to the ability to survive nearly complete protoplasmic dehydration and, in orthodox seeds, is acquired during maturation approximately halfway through the seed-filling phase. The presence of LEA (Late Embryogenesis Abundant) proteins correlates well with desiccation tolerance (Allagulova et al., 2003). The consistent correlation between desiccation tolerance in seeds and an accumulation of certain LEA proteins suggests that these proteins reduce desiccation-induced cellular damage (Blackman et al., 1995). Among LEA proteins, dehydrins (LEA, D-II family) have been the most commonly observed. Dehydrins belonging to different subclasses may accumulate to high levels in developing seeds during maturation (Lang and Palva, 1992; Goday et al., 1994). Some of the dehydrins, such as DHN-COG from pea, were found to accumulate in cotyledons in mid-to late embryogenesis, where severe dehydration conditions occur (Robertson and Chandler, 1994). Others, like DSP14 from Craterostigma plantagineum were present in drought-dehydrated plants, in all types of cells, but preferentially in phloem sieve tube elements in leaves and in embryonic cells in the seeds (Schneider et al., 1993). The tissue-specific localization and accumulation of SKn-type dehydrins in the vascular area may suggest that this type of dehydrin is required for the protection of mechanisms for water and nutrient transport to the rapidly dividing and growing cells of the apical part of organs. In cell dehydration conditions during seed maturation, they may protect macromolecules against the loss of water (Rorat, 2006).
Tracing the dehydration mechanism of maturing seeds in relation to morphology is expected to provide a better understanding of seed desiccation. Magnetic resonance imaging (MRI) is a non-destructive and non-invasive technique, which is useful for tracing water movement in plant tissues and for studying the biological implications in relation to water distribution, including seed germination (Fountain et al., 1998; Manz et al., 2005; Terskikh et al., 2005; Kikuchi et al., 2006; Garnczarska et al., 2007). However, this technique has not been effectively used for the determination of moisture distribution inside seeds during desiccation. Carrier et al. (1999) have used NMR imaging to study water content in developing white spruce seeds. Most studies published so far have been performed on seeds subjected to artificial drying, only mimicking physiological desiccation during maturation in planta (Kovács and Neményi, 1999; Ishida et al., 2004).
There is a lack of data on the mechanisms and route of water loss from seeds. The dehydration process of lupin seeds, especially the local inhomogeneities of water distribution inside the seed during maturation, have not been reported. In this study, the distribution of water in lupin seeds after harvesting at different seed stages was traced by MRI. To achieve a better understanding of the water state within the seed, changes in water status were also monitored in seeds during physiological desiccation in planta using NMR spectroscopy. Since the dehydration of seeds may be associated with the occurrence of LEA proteins, immunological analyses of dehydrin accumulation in seeds during maturation drying were performed.
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Materials and methods |
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Plant material
Seeds of yellow lupin (Lupinus luteus L., cv. Juno) were sown in the experimental fields of the Agricultural University of Pozna
at Zotniki (near Pozna, Poland). Irrigation, fertilization, and crop protection were designed to ensure optimal crop growth. Lupin pods were hand collected at 5 d intervals beginning at 15 d until 50 d after flowering (DAF), at the level of the second node in order to get pods of similar age. Seeds were removed from pods after harvest (three replicates of 15 seeds each) and used immediately in order to determine mean seed dry weight (mg) and seed moisture content (% FW) and water content calculated on a per seed basis. Freshly harvested seeds collected from 35–50 DAF at 5 d intervals were used for the analysis of water distribution and water state by MRI and NMR spectroscopy, respectively. Embryos isolated from seeds were also frozen in liquid nitrogen and used for immunological analysis. NMR analyses on germinating seeds were performed as described earlier by Garnczarska et al. (2007). In brief, seeds were soaked in water for the first 6 h and then transferred onto wet filter paper to support further imbibition. They were orientated on the filter paper with the hilum down. After the appropriate time (0, 0.25, 0.5, 0.75, 1, 1.25, 1.50, 1.75, 2, 4, 6, 12, and 24 h) a single seed was used for NMR relaxation time measurements. The estimated seed moisture contents were as follows: 9, 10, 21, 24, 31, 38, 39, 42, 52, 58, 63, 64, and 65%, respectively.
1H MRI and NMR spectroscopy
All magnetic resonance experiments reported here were carried out using an Avance DMX 400 MHz spectrometer (Bruker, Germany) equipped with a standard microimaging birdcage probehead. Freshly harvested seeds were placed in a glass tube – 15 mm outside diameter (Wilmad Glass Co., Inc., Buena, NJ, USA) – and oriented in the spectrometer probehead. For each developmental stage, five seeds were analysed. The angle between the long axis of seed and the direction of the B0 field was kept equal at 90°. All images of seeds were acquired using a spin echo pulse sequence (Callaghan, 1991) with a soft radio frequency (rf) pulses. Echo time TE, the time between a 90° rf pulse and spin echo origin, was equal to 6.62 ms. Repetition time TR, the time between successive experiments was set to 5 s. Because of the different water contents acquired by seeds in the course of dehydration, it was necessary to change the receiver gain of the spectrophotometer in order to restrict the signal dynamic and to optimize the signal/noise ratio of the images. The gain used covered the 26 dB range. Images were reconstructed on a 256x256 pixels matrix with Field of View (FOV) of 15 mm and slice thickness of 500 µm. For every seed three images were acquired in a perpendicular direction. All imaging experiments were performed at a temperature of 20 °C.
A spectrum for every seed was obtained by applying the Fourier Transform of the FID (Free Induction Decay) signal and measured with the following settings: repetition time (TR) 10 s, the hard 90° rf excitation pulse 12 µs. Transverse relaxation time T2 was measured using a standard spin echo Carr–Purcel–Meiboom–Gill (CPMG) (Meiboom and Gill, 1958) sequence with 4196 echoes. The time between echoes was 400 µs, but only even echoes were used in data analysis and therefore, the effective time between echoes was 800 µs. The experiment repetition time TR, likewise in the first case was 10 s. Rectangular 180° rf excitation pulses of 25 µs were used in the experiment. In both events, data were averaged eight times in order to improve the signal-to-noise ratio (SNR) and to allow for phase cycling. T2 determination was done five times for each developmental stage, and the mean values were calculated. Multi-exponential relaxation decay was analysed as described earlier (Garnczarska et al., 2007).
Protein extraction
Embryos (1 g) were ground in liquid nitrogen using a mortar and pestle. The powder was homogenized in 2 vols of 20 mM TRIS–HCl pH 7.5 with 5% (v/v)glycerol, 10 mM ß-mercaptoethanol and 35 µl protease inhibitor cocktail (Sigma). The homogenates were centrifuged at 13 000 g for 20 min. The supernatants were heated at 80 °C for 15 min followed by centrifugation at 13 000 g for 20 min. Protein measurement was performed according to Bradford (1976), using BSA as standard.
Electrophoresis and immunoblots
Proteins were separated by SDS-PAGE in 15% gels using a Mini-protean III cell (Bio-Rad, UK); 10 µg of total protein was loaded in each well. Separated polypeptides were transferred onto a PVDF membrane using a semi-dry transfer cell (Sigma-Aldrich, St Louis, MO, USA) for 60 min at 2 mA cm–2. After transfer, the membrane was blocked with 5% (w/v) dry non-fat milk in PBS with 0.05% Tween 20, for 1 h. After blocking the membrane was incubated overnight at 4 °C with the first antibody, a polyclonal antiserum raised against the dehydrin consensus polypeptide (EKKGIMDKIKEKLPG) (Stressgen) at a dilution of 1:1000 in PBS with 0.05% Tween 20 and 5% (w/v) dry non-fat milk. After three consecutive washes of 5 min each in PBS, the membrane was incubated for 2 h at room temperature with the secondary antibody, anti-rabbit IgG raised in goat and conjugated to biotin in PBS with 0.05% Tween 20 and 5% (w/v) dry non-fat milk. After three washes in PBS, blots were incubated for 15 min with alkaline phosphatase-conjugated streptavidin in PBS, followed by three washes with PBS. Polypeptides were revealed by incubating blots in BCIP/NBT. Independent protein extractions and immunoblots were performed in triplicate.
Tissue printing
Immunolocalization was performed by tissue printing on PVDF membrane. Coronal sections were performed through cotyledon tissues adjacent to the embryo axis using a sharp razor blade. The freshly cut surface was slightly blotted with absorbent paper to remove excess liquid. The tissue was then pressed against the membrane for 5 s, and the membrane was then dried at room temperature. At least three prints were prepared by repeated cutting close to the first cut from the same embryo. For each developmental stage, three embryos were analysed. The tissue-printed membrane was blocked by incubating it in 5% (w/v) dry non-fat milk in PBS with 0.05% Tween 20 for 1 h and treated with the first and secondary antibodies and developed as described for the immunoblots. Developed tissue prints were observed and photographed using a dissection microscope with a camera attachment (Canon).
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Seed development
Figure 1 shows the changes in dry weight and moisture content of lupin seeds during their development in planta. Seed moisture content declined steadily during the dry matter accumulation phase of seed development from 86% to 54% (fresh weight basis) at the time of physiological maturity, but then declined much more rapidly during the next 5 d (Fig. 1A). The final moisture content was about 15% at 50 DAF. However, the water content (expressed on a per seed basis) increased until 30 DAF and decreased thereafter (Table 1). A ratio of water content and dry weight decreased gradually from 15 DAF to 45 DAF. After 45 DAF water and dry weight ratio remained essentially constant (Table 1). Physiological maturity, as indicated by maximum seed dry weight, relative water content below 55% and loss of green colour from embryo tissue, occurred at around 40 DAF. Dry weight of the seed increased following the sigmoidal curve (Fig. 1B).
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Spatial distribution of water protons
MR imaging was used to measure the spatial distribution of water in maturing lupin seeds from 35–50 DAF (Fig. 2). MR images were obtained in three orthogonal orientations: axial, or transverse, in which the plane is perpendicular to the embryonic axis and two longitudinal planes, coronal and sagittal which are parallel to the embryonic axis and orthogonal to each other. The images presented in Fig. 2 are limited to one 2D superficial and median or near-median slice in the sagittal, coronal and axial plane, respectively. The slices were taken as a series of sections from single seed at a given time. In lupin seed collected at 35 DAF (approximately 67% water per FW) the most intense signal is observed in seed coat and the outer part of cotyledons in the hilum area. MR images reveal in the axis a central zone with stronger signal which corresponds to vascular bundle, enclosed by a concentric zone of lower intensity signal which probably corresponds to the cortex. In the central plane of cotyledons a vascular network is discernible. As the dehydration progresses the seed is getting smaller and the differences in the intensity of the signal between cotyledons and embryonic axis disappear. In the mature lupin seed (50 DAF) the water content is very low (15% water per FW) but the seed is clearly visible in all planes. Both cotyledons and embryonic axis show the same intensity of the signal, however, in contrast to the earlier stages, the outer parts of cotyledons seem to be less hydrated. The void between cotyledons is seen as the area with low water signal. An interesting feature of lupin seed at 50 DAF is the presence of two areas (seen as dots) with the highest water signal easily distinguished in all planes. The same signal appeared in seeds collected at 40 DAF and 45 DAF and refers to vascular bundles seen in the coronal plane in the seed 40 DAF.
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CPMG experiment
The overall water loss from the seed, measured as the moisture content, does not bring much light on water compartmentalization or distribution in a seed's structure. Therefore, the CPMG transverse relaxation time experiment was applied for each seed as a function of developmental stage (Fig. 3) and seed moisture content (Fig. 4), respectively. The later one is presented together with data obtained for germinating lupin seeds. The time scale of seed development, as in MRI experiments, was from 35–50 DAF. All decays of the spin-echo amplitude registered over three decades in the CPMG experiment were non- or multi- exponential, indicating the coexistence of different states of water inside the seed. These decays acquired for each developmental stage or seed moisture content were best fitted using a three exponent model, with six free fitting parameters allowing for water diversity characterized by three values of T2 time. Moreover, the abundance or population of the water for each of the three components was also determined through the fit. A significant decrease in T2 value of the component with low molecular mobility was noticed in seeds collected at 45 DAF and 50 DAF (Fig. 3A). The contribution of different water fractions remained almost stable during maturation, but in seeds harvested at 50 DAF the complex water exchange process between components inside the lupin seed was observed (Fig. 3B). The decrease of the component with low molecular mobility was accompanied by an increase of water fractions with longer relaxation times. Figure 4 presents a comparative analysis of components of spin–spin relaxation (T2) both in maturing and germinating lupin seeds expressed versus seed moisture content. For all water components, values of T2 from maturing seeds overlap values obtained for germinating seeds, indicating that the same water fractions are observed in lupin seeds at both physiological processes, i.e. in hydrating and desiccating seeds with the same water content. However, the contribution of water fractions showing higher molecular mobility decreases at the completion of seed maturation compared with germinating seeds (Fig. 4B).
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Accumulation of dehydrin
To study the pattern of dehydrin accumulation during lupin seed development, total soluble heat stable proteins extracted from embryos collected from different seed stages were separated by electrophoresis and immunoblotted (Fig. 5). The anti-dehydrin antiserum reacted with three polypeptides isolated from lupin embryos with relative molecular masses of 73, 43, and 28 kDa, respectively. The polypeptide with molecular mass of 43 kDa was not detectable in lupin embryos isolated at 35 DAF, but appeared in the course of dehydration. The temporal pattern of dehydrin protein accumulation correlates well with seed maturation.
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Immunolocalization of dehydrin protein
Tissue prints were prepared using lupin embryos collected at 40 DAF. Tissue prints were analysed using the anti-dehydrin serum. Anti-dehydrin serum showed strong recognition associated with vascular tissue (Fig. 6A). Similar results were obtained for lupin embryos collected at 45 DAF (data not shown). It was not possible to obtain impressions of younger embryos, because the softness and high water content of these tissues precluded clear impressions. Dry mature seeds were too hard to obtain impressions when pressed onto the PVDF membranes. To control for non-specific reaction of secondary antibody with tissue prints, tissue printing not incubated with a primary antibody (Fig. 6B) or without a secondary antibody (Fig. 6C) were made and just showed a light background.
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Discussion |
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Magnetic resonance imaging visualized the process of water loss in maturing lupin seeds. MRI showed local inhomogeneities of water distribution inside the seed at the late stage of seed filling and afterwards during maturation drying. In seeds harvested at 35 DAF the highest 1H-NMR signal was observed in the seed coat and the outer part of the cotyledons, especially in the hilum area, which enables the contact of the seed with maternal tissues (Fig. 2). The hilum is the site of water uptake during imbibition (Garnczarska et al., 2007), indicating that it is readily permeable to water. Seeds gain water during development and then lose water during maturation (Table 1). Water content declined during seed filling as the dry weight of the seed accumulated (Fig. 1). This developmental change in seed water content is largely independent of the plant or seed water potential, since it is due to the water volume being displaced from the expanded cells by the water deposition of reserves within the storage tissues. The embryonic cotyledons are the ultimate sink for photosynthate imported from the maternal tissues (Thorne, 1981; Patrick and Offler, 2001). Transport of solutes and water into seeds is primarily through the phloem. During the dry weight accumulation phase, phloem transport, at least, must remain intact and functional between the mother plant and the unloading tissues in the seed coat. Nutrients and water exit the importing phloem by bulk flow. Water returns to the parent plant in the xylem, while nutrient loss is prevented by selectively-permeable apoplasmic barriers and xylem discontinuties (Patrick and Offler, 2001). In seeds, both import of solutes and recycling of water need to occur simultaneously (Bradford, 1994). In developing legume fruits, maximum water loss by transpiration occurs during seed filling (Pate et al., 1977), although the fruits have low stomatal frequencies (Shackel and Turner, 2000). NMR images of 35 DAF lupin seeds showing the highest 1H-NMR signal in the hilum area confirm the involvement of vascular bundles of the funiculus in the translocation of water between the pod and the seed coat. However, NMR imaging used in this study does not allow the resultant direction of water movement to be determined. At around the time of physiological maturity (40 DAF) and afterwards, the abrupt reduction in seed moisture content (Fig. 1) and MR image intensity (Fig. 2) may correspond to an interruption of water supply to the pod. The pod begins rapid senescence (data not shown) because the interruption of the vascular connection with the mother plant, accompanied by fruit transpiration, creates a severe water deficit (Le Deunff and Rachidian, 1988). Funiculus detachment and senescence of the pod cut off the water supply to the seed. The hilum is where the seed will eventually abscise from the funiculus. Seeds not receiving water through the phloem rapidly dehydrate so, at the completion of seed maturation, water loss is associated with actual tissue dehydration to air dryness (Fig. 1). During this phase, there is a net loss of water from the seed (Adams and Rinne, 1979). All organs of the seed dehydrate. Since the seed coat is the tissue through which photosynthates are transported to the embryo this organ is principally involved in desiccation. MR images of seeds collected at 45 DAF and 50 DAF show that, in the outer parts of the seed, the decline of the 1H-NMR signal intensities were faster than those of the central part of the seed (Fig. 2). Water seemed to evaporate from the seed surface. The same mechanism of water loss was observed in rice seeds (Ishida et al., 2004).
Water content is a poor indicator of the physiological status of water in developing seeds, since it is changing continuously due to dry weight accumulation and seed dehydration and does not provide information on the availability of water and its fractions. 1H-NMR spectroscopic measurements on maturing lupin seeds revealed three populations of protons, each with a different magnetic environment that causes a different relaxation rate (Fig. 3). These populations may correspond to water molecules differing in mobility. To identify water fractions, the general pattern of occurrence of different water components appearing during seed maturation was compared with water fractions observed in germinating seeds with regard to moisture content at both the physiological stages (Fig. 4). Interestingly, lupin embryo tissues showed the same water fractions at the same moisture content during both seed development and germination. NMR studies on hydrated seeds suggest that protons with short relaxation time are associated with bound/structural water, protons of medium relaxation time are associated with intracellular/cytoplasmic water, and protons of long relaxation time are associated with extracellular water (Isobe et al., 1999; Krishnan et al., 2004; Nagarajan et al., 2005; Garnczarska et al., 2007). The results of transverse relaxation time analyses indicate that the contribution of different water fractions remained constant in the course of seed maturation, although the seeds significantly shrank in size and water content decreased markedly. The only exception is the seed harvested at 50 DAF showing the complex water exchange process between components inside the seed (Fig. 3B). A significant decrease in the T2 value of the component with lower mobility (Fig. 3A) indicates that immobilization of water molecules by macromolecules (carbohydrates, proteins, etc.) within the seed matrix becomes more effective in the course of desiccation time. According to Hazlewood et al. (1974), interaction between biopolymer surface and water reduce the motion of water molecules that shortens the relaxation time. The pattern of components of spin–spin relaxation T2 and the contribution of various T2 relaxation time components of water during seed maturation followed the pattern inversely related to seed germination (Fig. 4). Only in seeds harvested at 45 DAF and 50 DAF was the contribution of water fractions corresponding to intracellular and extracellular water lower in maturing seeds compared with germinating seeds. It may be the consequence of a reduction of these water fractions through intensive evaporation from the seed surface. It is generally accepted in seed physiology that seed development and germination are two distinctly different processes. Seed development and maturation are associated with an overall loss of moisture. In lupin seed, moisture decreased from 86% to 15% (Fig. 1). Germination has an inverted moisture regime compared with that of development. However, studies that show biochemical similarities between seed development and germination do exist. For example, Turley and Trelease (1990) and Modi et al. (2001) showed the presence of post-germinative respiration enzymes in cotton and soybean seed, respectively, during maturation. Bewley (1997) reported accounts of biochemical events, including hormone occurrence, during seed development and germination.
Since the dehydration of seeds may be associated with the occurrence of dehydrin proteins, immunological analyses of dehydrin accumulation in lupin embryos isolated from seeds from 35–50 DAF were performed. Western blots show that dehydrin protein of approximately 43 kDa was not detectable in 35 DAF lupin embryos (Fig. 5), but appeared when seeds reached physiological maturity and the moisture content was below 55%. The accumulation of three detected dehydrins observed by western blot analysis correlates well with seed maturation. The fact of intensive synthesis and accumulation of LEA proteins in seeds at the late stages of their maturation, during dehydration, suggests the involvement of these proteins in protective reactions promoting maintenance of embryo structures under conditions of water deficit (Allagulova et al., 2003). The barley dehydrin DHN12 gene is specifically expressed during the embryonic stage of development (i.e. is not expressed in vegetative tissues even under stress conditions (Choi and Close, 2000). The amino acid composition of dehydrins is characterized by the high content of charged and polar residues, and this determines their biochemical properties including thermostability. This may promote their specific protective functions under conditions of cell dehydration: dehydrins may prevent the coagulation of macromolecules and maintain the integrity of crucial cell structures (Allagulova et al., 2003; Rorat, 2006).
In lupin seeds harvested at 40 DAF and afterwards the highest 1H-NMR signal was observed in cotyledons tissue adjacent to the embryo axis (Fig. 2). The same signal was observed in germinating lupin seeds and light microscopy observations confirmed the presence of vascular bundles in this area (Garnczarska et al., 2007). Immunolocalization by tissue printing showed dehydrin proteins to be associated with vascular tissues (Fig. 6). The localization of dehydrin-like protein in vascular tissues was also observed in barley under cold acclimation (Bravo et al., 1999). Vascular localization of dehydrins might promote water influx into the vascular bundles. The vascular system of cotyledons is crucial for the translocation of seed reserves from the cotyledons to the growing axis during seed germination, and it therefore seems likely that the localization of dehydrins in these parts of the cotyledons is required for the protection of mechanisms for water and nutrient transport to the dividing and growing cells of the embryonic axis. Dehydrins might function as water attractants during the transport of water and seed reserves to sink tissue (Rorat, 2006). Water retained in the vascular bundles of dry seeds may also protect them from imbibitional injury. The areas within dry seed with elevated water concentration had higher water uptake capacities during imbibition compared with other tissues as shown by Manz et al. (2005). According to Vertucci and Leopold (1984) tissues could be somewhat protected from imbibitional injury by elevating the initial moisture level.
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The applicability of MR imaging and NMR spectroscopy to study seed dehydration during maturation in planta has been demonstrated. This paper, visualizing water distribution and water status in maturing lupin seeds, makes a convincing contribution to a better understanding of water loss events in leguminous seeds. Comparative analysis of lupin seeds from individual developmental stages with germinating seeds of a similar water content shows that, at a given moisture content, the same water components are observed in developing and germinating seeds. It seems that it is possible to identify related stages of seed development and germination on the basis of common water status, although the mechanisms by which the water content and dry matter increase/decrease in developing seeds are different from those in germinating seeds.
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Acknowledgements |
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This work was supported by the State Committee for Scientific Research (KBN), grant 2 P06R 085 26.
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References |
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