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A comparative study of water distribution and dehydrin protein localization in maturing pea seeds
ARTICLE IN PRESS Journal of Plant Physiology 165 (2008) 1940—1946
www.elsevier.de/jplph
A comparative study of water distribution and dehydrin protein localization in maturing pea seeds Małgorzata Garnczarskaa,, Tomasz Zalewskib, Łukasz Wojtylaa a
´, Poland Department of Plant Physiology, A. Mickiewicz University, Umultowska 89, 61-614 Poznan ´, Poland Department of Macromolecular Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznan
b
Received 26 February 2008; received in revised form 25 April 2008; accepted 27 April 2008
KEYWORDS Dehydrins; MRI; Pisum sativum; Seed maturation; Water content
Summary In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35 kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles. & 2008 Elsevier GmbH. All rights reserved.
Introduction A large group of seeds, the so-called ‘‘orthodox’’ seeds, are desiccation tolerant; they are able to survive in a dry state and germinate after prolonged periods. Thus, the acquisition of desiccation Corresponding author. Tel.: +48 61 829 5894;
fax: +48 61 829 5890. E-mail address: [email protected] (M. Garnczarska).
tolerance is an important process during seed development. Seed development can be divided into three stages (Kermode and Finch-Savage, 2002). The first is characterized by cell division and histodifferentiation. Moisture content during this phase is high and stable. In the second phase – seed filling – accumulation of macromolecules such as storage proteins, lipid and starch occurs, and there is also a slight decrease in the moisture content. Seed filling is terminated when the seed
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.04.016
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds reaches maximum dry matter accumulation. Desiccation tolerance is acquired at the stage of maximum dry matter accumulation, or a few days earlier when the water potential of the developing embryo is lowered, usually in response to a natural severing of the vascular connection to the mother plant, but also from premature harvest (Adams and Rinne, 1981; Blackman et al., 1992). At the end of seed dehydration, the moisture content decreases even below 10% (Leprince et al., 1993). Acquisition of desiccation tolerance of orthodox seeds is associated with accumulation of di- and oligosaccharides (Leprince et al., 1993; Bailly et al., 2001; Hoekstra et al., 2001), synthesis of late embryogenesis abundant (LEA) protein (Close, 1997; Farrant et al., 2004), heat-shock proteins (Wehmeyer et al., 1996) and activation of antioxidative defenses (Bailly et al., 2001; Bailly, 2004). LEA proteins are produced during seed development, and their expression is linked to the acquisition of desiccation tolerance, but the induction of their synthesis also occurs during cold or osmotic stress (Welin et al., 1994). Although their precise function and catalytic activity are unknown, they are assumed to protect cellular or molecular structures from the damaging effects of water loss (Goyal et al., 2005). LEA group 2 proteins called dehydrins (DHNs) are of particular interest in this respect. They are characterized by an expression pattern typical of LEA proteins and seem to protect cells against dehydration by maintaining the structure of proteins and water binding (Close, 1996, 1997; Farrant et al., 2004). During drying after maximum dry matter accumulation, the seed is prepared to survive in adverse weather conditions by reducing the water content inside seed tissues. Such dehydration rates might have harmful effects on cells, tissues and organs, and so protective events must also take place. Recent data indicate that DHNs may play a protective role during dehydration. In this study, we investigated water distribution during seed maturation drying in comparison with spatial and temporal accumulation of DHNs inside Pisum sativum seeds.
1941 15 to 40 d after flowering (DAF). Seeds were removed from pods after harvest and used immediately in order to determine mean seed fresh and dry mass (mg) and seed water content expressed on a dry mass basis (g H2O g 1DM). Freshly harvested seeds collected from 30 to 45 DAF were used for analysis of water distribution by magnetic resonance imaging (MRI). Embryos isolated from seeds were also frozen in liquid nitrogen and used for immunological analysis. Protein extraction Embryos (1 g) were ground with liquid nitrogen, and the powder was homogenized on ice in 2 vols of 20 mM Tris–HCl buffer, pH 7.5, with 5% (v/v) glycerol, 10 mM bmercaptoethanol and 35 mL protease inhibitor cocktail (Sigma) for 1 h. After that time, the homogenates were centrifuged at 13,000g for 20 min. The supernatants were collected and heated at 80 1C for 15 min. After cooling on ice, the probes were centrifuged at 13,000g for 20 min, and the supernatants were taken for further analysis. Protein measurement was performed according to Bradford (1976), using BSA as standard. Electrophoresis and immunodetection of dehydrins Samples of extracts were electrophoresed in 15% polyacrylamide SDS slab gels using a Mini-protean III cell (Bio-Rad, UK). In all, 10 mg of total protein was loaded in each well. Electrophoresis-separated proteins were then transferred onto a PVDF membrane (Immobilon P) using a semi-dry transfer system (Sigma-Aldrich, St Louis, MO, USA). The transfer was carried out at 2 mA for each square cm for 60 min. The membrane was blocked in phosphate buffer saline (PBS) with 5% (w/v) skimmed milk (SM) for 1 h. After blocking, the membrane was incubated overnight at 4 1C with primary anti-DHN antibody (Stressgen Bioreagents Corp., Victoria, Canada) in PBS with 5% SM and 0.05% Tween 20. After three consecutive washes of 5 min each in PBS, the membrane was incubated with biotinylated secondary anti-rabbit antibody (Sigma) in PBS with 5% SM and 0.05% Tween 20 for 2 h. After three washes in PBS, the membrane was incubated for 15 min with streptavidin conjugated with alkaline phosphatase in PBS followed by three washes with PBS. The visualization of DHN protein was carried out with BCIP/NBT (Sigma Fast). Independent protein extractions and immunoblots were performed in triplicate. Tissue printing
Material and methods Plant material Seeds of pea (P. satium L., cv. Piast) were sown in the experimental fields of the Agricultural University of Poznan ´ at Złotniki (near Poznan ´, Poland). Irrigation, fertilization and crop protection were designed to ensure optimal crop growth. Pea pods were hand collected from
Transverse sections of pea cotyledons and embryo axes were made in the median or near-median part of the seeds with a razor blade. The freshly cut surface was briefly wiped on Whatman paper to remove excess of liquid. The tissue was then pressed against the PVDF membrane for approximately 5–10 s, and the membrane was dried at room temperature. The tissue-printed membrane was blocked by incubating in 5% (w/v) SM in
ARTICLE IN PRESS 1942 PBS buffer for 1 h and treated for immunodetection of DHNs as described above. 1
H MRI
Experiments reported here were carried out using an Avance DMX 400 MHz spectrometer (Bruker, Germany) equipped with a standard microimaging birdcage probe head. 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 probe head. For each developmental stage, five seeds were analyzed. The angle between the long axis of the seed and the direction of the B0 field was 901. All images of seeds were acquired using a spin echo pulse sequence (Callaghan, 1991) with soft radio frequency (rf) pulses. Echo time TE, the time between a 901 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 different water content 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 optimize the signal/noise ratio of images. The gain used covered the 26 dB range. Images were reconstructed on a 256 256 pixel matrix with a field of view of 15 mm and slice thickness of 500 mm. Three images were acquired in a perpendicular direction for every seed. All imaging experiments were performed at a temperature of 20 1C.
Results Seed development The changes in fresh mass, dry mass and water mass (expressed on a per seed basis) of pea seeds during their development in planta are presented
M. Garnczarska et al. in Figure 1. An increase in fresh mass occurred from 15 to 30 DAF, while water mass increased until 30 DAF and decreased thereafter. The final water mass was about 45 mg per seed at 45 DAF. Dry mass of the seed increased following a sigmoidal curve. The maximum dry matter accumulation occurred at around 35 DAF.
Magnetic resonance imaging MRI was used to measure the spatial distribution of water in maturing pea seeds from 30 to 45 DAF (Figure 2). MR images were obtained in two orthogonal orientations: axial, or transverse, in which the plane is perpendicular to the embryonic axis; and sagittal, which is parallel to the embryonic axis. The images presented in Figure 2 are limited to one 2D median or near-median slice in the sagittal and axial planes, respectively. The slices were taken as a series of sections from a single seed at a given time. In pea seeds collected at 30 DAF (approximately 1.28 g H2O g 1DM), the most intense signal was observed in the seed coat and the outer part of cotyledons. The signal coming from water was also strong in the vascular network inside cotyledons. In pea seeds at 35 DAF, the water content decreased to 0.98 g H2O g 1DM and the outer parts of seed were characterized by lower 1HNMR signals compared to seeds harvested at 30 DAF. Images of water distribution in pea seeds at 40 DAF showed that the embryo axis was more hydrated than cotyledons, and the outer parts of cotyledons were the areas with more intense signals than the inner parts. The vascular network was still well distinguished. At 45 DAF, the water content was very low and the 1H-NMR signal faded away, and traces of water could only be seen in embryo axis.
Accumulation of dehydrins
Figure 1. Changes in dry, fresh and water mass of pea seeds during their development. Means of three replicates of 15 seeds each 7SD.
Accumulation of DHN proteins was investigated using an immunochemical reaction with an antibody against the DHN C-terminal synthetic peptide based on a DHN consensus sequence (K-segment), with an amino-terminal cysteine (C) residue added (Stressgen). Total soluble heat-stable proteins extracted from pea embryos collected from different seed stages were separated by electrophoresis and immunoblotted. The analysis revealed three polypeptides with molecular masses of 29, 30 and 35 kDa (Figure 3). All polypeptides were present in seeds collected at 15 DAF, but the abundance of DHNs proteins with molecular masses of 30 and 35 kDa increased during maturation. The amount of
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds
1943
Figure 2. MR images of maturing pea seeds from 30 to 45 DAF. Pictures show median or near-median slice in the sagittal (the first row) and axial plane (the second row). Numbers indicate days after flowering. Images were chosen among five views and were selected for being representative of all of the views.
Figure 3. Dehydrins in maturing pea embryos from 30 to 45 DAF. Heat-stable soluble proteins were extracted and 10 mg of protein per lane was loaded on the gel. Dehydrins were detected by western blotting with antiserum raised against the dehydrin consensus polypeptide comprised of residues (C)TGEKKGIMDKIKEKLPGQH (Stressgen). Independent protein extractions and immunoblots were performed in triplicate and yielded identical results.
29 kDa polypeptide decreased slightly during maturation drying. To control for nonspecific reaction of antibodies, blots not incubated with a primary antibody or without a secondary antibody were made and showed no signal. The control reactions without primary and secondary antibodies and streptavidin conjugated with alkaline phosphatase eliminated the possibility of biotinylated proteins having been observed.
Immunolocalization of dehydrins Localization of DHNs in seeds was determined by tissue printing on a PVDF membrane. Tissue
Figure 4. (A) Protovascular localization of dehydrin protein in axial sections performed through pea cotyledons and embryo axis from seed collected at 30 DAF. The experiment was carried our three times with similar results. (B) 1H-NMR signal in axial section of pea seed collected at 30 DAF. axis, embryo axis; cot, cotyledons.
printings were analyzed using the anti-DHN serum. Figure 4 presents tissue prints of seeds collected at 30 DAF. Similar results were obtained with seeds collected at 35 DAF (data not shown). Anti-DHN serum showed strong recognition associated with protovascular tissue, both in the embryo axis and cotyledons. It was not possible to obtain impressions of younger embryos, because the softness and high water content of these tissues precluded clear impressions. It was also too difficult to obtain impressions of dry mature seeds by pressing onto the PVDF membranes.
ARTICLE IN PRESS 1944
Discussion The development of orthodox seeds is terminated by maturation and desiccation, which result in gradual reduction in metabolism as water is lost from seed tissues and the embryo passes into a metabolically inactive or quiescent state (Kermode and Finch-Savage, 2002). Water mass declined during pea seed filling as the dry mass of the seed accumulated (Figure 1). This change in water content was due to water volume being displaced from the expanded cells by the water deposition of reserves within the storage tissues. During desiccation, dry mass accumulation slowed, and fresh mass declined markedly and was accompanied by a rapid decline in water mass (Figure 1). During this phase, there was a net loss of water from the seed. MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside pea seeds (Figure 2). In seeds harvested at 30, 35 and 40 DAF, the intensity of the signal coming from water declined from the inner part of cotyledons tissues. The inhomogeneity of water distribution in pea seeds may be correlated with the gradient of storage substances accumulation within cotyledons. Most seeds lose water as reserves are deposited primarily within storing tissues, displacing water from the cells. In pea seeds, starch can comprise around 50% of the dry mass (Go ´recki, 2001). Starch accumulation begins in parenchyma cells at the inner adaxial region of the cotyledons and spreads to the abaxial region (Borisjuk et al., 1995, 2003). The pattern of water loss may reflect the pattern of starch deposition in cotyledons. MR images of lupine seeds showed that, in contrast to pea seeds, the decline of the 1H-NMR signal intensities was more rapid in the outer parts of the seed than in the central part (Garnczarska et al., 2007) and water appeared to evaporate from the seed surface. The differences in the route and mechanism of water loss between pea and lupine may be associated with differences in seed reserves composition, since proteins constitute up to 45% of the DM of lupine seed and starch represents only up to 2% of the DM of the seed (data not shown). However, this may not be only because of differences in water-absorbing capacity (i.e. hydroscopic properties) of pea and lupine, since barriers to water movement might also influence the drying rate of seeds. At the end of the seed filling stage, the abrupt reduction in pea seed water mass and MR image intensity may also correspond to an interruption of water supply to the pod as well as the senescence of the pod. During drying after maximum dry matter accumulation, the differ-
M. Garnczarska et al. ences in the intensity of the signals between cotyledons and the embryonic axis became apparent. In seeds harvested at 40 DAF, the embryonic axis appeared to be more hydrated than cotyledons in contrast to earlier stages, and even in dry mature seeds traces of water could be seen in the axis. One explanation is that dry matter accumulated in reserve-storing tissues does not have the same hydroscopic properties as materials in the embryonic axes. It can be a part of the protective mechanism since, according to Vertucci and Leopold (1984), tissues could be somewhat protected from imbibitional injury by elevating the initial moisture level. The 1HNMR 2D micro-imaging of low-hydrated tobacco seeds also showed that there were localized tissue areas in the covering layers with higher proton mobility (Leubner-Metzger, 2005). This finding indicates the presence of free water in air-dry seeds. Proton NMR indicated retention of mobile water during dehydration in the desiccation-tolerant grass Eragrostic nindensis (Balsamo et al., 2005) as part of the strategy for surviving water loss in vegetative tissues. Drying after maximum dry matter accumulation is either coincident with or subsequent to the acquisition of desiccation tolerance. It has been demonstrated that DHNs, a group-2 LEA class of proteins, accumulate under conditions of water deficit (Close, 1997; Farrant et al., 2004). They are expected to play a protective role during dehydration; however, functional evidence is scare. Immunological analyses of DHN accumulation were performed in pea embryos isolated from seeds from 30 to 45 DAF. Western blots showed that the temporal pattern of accumulation of two DHNs with molecular masses of 30 and 35 kDa correlated well with seed maturation (Figure 3). This suggests the involvement of these proteins in protective reactions promoting maintenance of embryo structures under conditions of water deficit (Allagulova et al., 2003). The amino acid composition of DHNs with high content of charged and polar residues may promote their specific protective functions under conditions of cell dehydration (Allagulova et al., 2003; Rorat, 2006). Immunolocalization by tissue printing showed DHN proteins to be associated with protovascular tissues of both the embryo axis and cotyledons (Figure 4). This pattern of DHNs localization corresponds well with water distribution inside pea seeds. The intense 1H-NMR signal appeared in seeds collected at 35 DAF, and referred to protovascular bundles. As shown by Bauby et al. (2007), during Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell division, giving rise to vascular precursors cells
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds (protophloem and protoxylem precursors) in developing organs. DHNs present in the embryonic axis may increase its water-absorption capacity. The localization of DHN protein in vascular tissues was also observed in maturing lupine embryos (Garnczarska et al., 2007) and in barley under cold acclimation (Bravo et al., 1999). DHNs are highly hydrophilic proteins, which might explain the more intense signal coming from water in MR images in the vascular bundle region inside seeds. Vascular localization of DHNs might promote water influx into the vascular bundles. DHNs may function as water attractants during the transport of water and seed reserves to sink tissue (Rorat, 2006).
Conclusions The loss of the water signal in maturing pea seeds proceeds gradually from the inner to the outer part of cotyledons and is likely orientated in the same direction as starch accumulation. The pattern of DHN localization reflects the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.
Acknowledgment This work was supported by the State Committee for Scientific Research (KBN), Grant 2 P06R 085 26.
References Adams CA, Rinne RW. Seed maturation in soybeans (Glycine max L. Merr) is independent of seed mass and the parent plant, yet is necessary for production of viable seeds. J Exp Bot 1981;32:615–20. Allagulova CR, Gimalov FR, Shakirova FM, Vakhitow VA. The plant dehydrins: structure and putative functions. Biochemistry 2003;68:1157–65. Bailly C. Active oxygen species and antioxidants in seed biology. Seed Sci Res 2004;14:93–107. Bailly C, Audigier C, Ladonne F, Wagner MH, Coste F, Corbineau F, et al. Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality. J Exp Bot 2001;52:701–8. Balsamo RA, Willigen CV, Boyko W, Farrant J. Retention of mobile water during dehydration in the desiccationtolerant grass Eragrostis nindensis. Physiol Plant 2005;124:336–42. Bauby H, Divol F, Truernit E, Grandjean O, Palauqui JC. Protophloem differentiation in early Arabidopsis
1945 thaliana development. Plant Cell Physiol 2007;48: 97–109. Blackman SA, Obendorf RL, Leopold AC. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol 1992;100: 225–30. Borisjuk L, Rolletschek H, Wobus U, Weber H. Differentiation of legume cotyledons as related to metabolic gradients and assimilate transport into seeds. J Exp Bot 2003;54:503–12. Borisjuk L, Weber H, Panitz R, Manteuffel R, Wobus U. Embryogenesis of Vicia faba L.: histodifferentiation in relation to starch and storage protein synthesis. J Plant Physiol 1995;147:203–18. Bradford M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. Bravo LA, Close TJ, Corcuera LJ, Guy CL. Characterization of an 80-kDa dehydrin-like protein in barley responsive to cold acclimation. Physiol Plant 1999; 106:177–83. Callaghan PT. Principles of nuclear magnetic resonance microscopy. Oxford: Oxford University Press; 1991. Close TJ. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 1996;97:795–803. Close TJ. Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiol Plant 1997;1544:196–206. Farrant JM, Bailly C, Leymarie J, Hamman B, Come D, Corbineau F. Wheat seedlings as a model to understand desiccation tolerance and sensitivity. Physiol Plant 2004;120:563–74. Garnczarska M, Zalewski T, Kempka M. Changes in water status and water distribution in maturing lupin seeds studied by MR imaging and NMR spectroscopy. J Exp Bot 2007;58:3961–9. Goyal K, Walton LJ, Tunnacliffe A. LEA proteins prevent protein aggregation due to water stress. Biochem J 2005;388:151–7. Go ´recki RJ. Seed physiology and biochemistry. In: Hedley CK, editor. Carbohydrates in grain legume seeds: improving nutritional quality and agronomic characteristic. CABI Publishing; 2001. p. 117–44. Hoekstra FA, Golovina EA, Buitink J. Mechanisms of plant desiccation tolerance. Trends Plant Sci 2001;6: 431–8. Kermode AR, Finch-Savage BE. Desiccation sensitivity in orthodox and recalcitrant seeds in relation to development. In: Black M, Pritchard HW, editors. Desiccation and survival in plants. Drying without dying. CABI Publishing; 2002. p. 150–74. Leprince O, Hendry GAF, McKersie BD. The mechanisms of desiccation tolerance in developing seeds. Seed Sci Res 1993;3:231–46. Leubner-Metzger G. b-1,3-glucanase gene expression in low-hydrated seeds as a mechanism for dormancy release during tobacco after-ripening. Plant J 2005;41:133–45.
ARTICLE IN PRESS 1946 Rorat T. Plant dehydrins – tissue location, structure and function. Cell Mol Biol Lett 2006;11:536–56. Vertucci CW, Leopold AC. Bound water in soybean seed and its relation to respiration and imbibitional damage. Plant Physiol 1984;75:114–7. Wehmeyer N, Hernandez LD, Finkelstein RR, Vierling E. Synthesis of small heat-shock protein is part of the
M. Garnczarska et al. developmental program of late seed maturation. Plant Physiol 1996;112:747–57. Welin BV, Olson A, Nylander M, Palva ET. Characterization and differential expression of dhn/lea/rablike genes during cold acclimation and drought stress in Arabidopsis thaliana. Plant Mol Biol 1994;26: 131–44.
www.elsevier.de/jplph
A comparative study of water distribution and dehydrin protein localization in maturing pea seeds Małgorzata Garnczarskaa,, Tomasz Zalewskib, Łukasz Wojtylaa a
´, Poland Department of Plant Physiology, A. Mickiewicz University, Umultowska 89, 61-614 Poznan ´, Poland Department of Macromolecular Physics, A. Mickiewicz University, Umultowska 85, 61-614 Poznan
b
Received 26 February 2008; received in revised form 25 April 2008; accepted 27 April 2008
KEYWORDS Dehydrins; MRI; Pisum sativum; Seed maturation; Water content
Summary In this study, the distribution of water in pea seeds after harvesting at different seed stages was traced by magnetic resonance imaging (MRI). MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside seeds. The intensity of the signal coming from water declined from the inner to the outer part of cotyledon tissue. This spatial inhomogeneity of water signals inside cotyledons may be correlated with the gradient of storage substances accumulation within cotyledons. Tissue localization of dehydrins showed the presence of dehydrin protein in the area of protovascular tissue of both the embryo axis and cotyledons. The temporal accumulation of two dehydrin proteins with molecular masses of 30 and 35 kDa correlated well with seed desiccation. The pattern of dehydrin localization reflected the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles. & 2008 Elsevier GmbH. All rights reserved.
Introduction A large group of seeds, the so-called ‘‘orthodox’’ seeds, are desiccation tolerant; they are able to survive in a dry state and germinate after prolonged periods. Thus, the acquisition of desiccation Corresponding author. Tel.: +48 61 829 5894;
fax: +48 61 829 5890. E-mail address: [email protected] (M. Garnczarska).
tolerance is an important process during seed development. Seed development can be divided into three stages (Kermode and Finch-Savage, 2002). The first is characterized by cell division and histodifferentiation. Moisture content during this phase is high and stable. In the second phase – seed filling – accumulation of macromolecules such as storage proteins, lipid and starch occurs, and there is also a slight decrease in the moisture content. Seed filling is terminated when the seed
0176-1617/$ - see front matter & 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2008.04.016
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds reaches maximum dry matter accumulation. Desiccation tolerance is acquired at the stage of maximum dry matter accumulation, or a few days earlier when the water potential of the developing embryo is lowered, usually in response to a natural severing of the vascular connection to the mother plant, but also from premature harvest (Adams and Rinne, 1981; Blackman et al., 1992). At the end of seed dehydration, the moisture content decreases even below 10% (Leprince et al., 1993). Acquisition of desiccation tolerance of orthodox seeds is associated with accumulation of di- and oligosaccharides (Leprince et al., 1993; Bailly et al., 2001; Hoekstra et al., 2001), synthesis of late embryogenesis abundant (LEA) protein (Close, 1997; Farrant et al., 2004), heat-shock proteins (Wehmeyer et al., 1996) and activation of antioxidative defenses (Bailly et al., 2001; Bailly, 2004). LEA proteins are produced during seed development, and their expression is linked to the acquisition of desiccation tolerance, but the induction of their synthesis also occurs during cold or osmotic stress (Welin et al., 1994). Although their precise function and catalytic activity are unknown, they are assumed to protect cellular or molecular structures from the damaging effects of water loss (Goyal et al., 2005). LEA group 2 proteins called dehydrins (DHNs) are of particular interest in this respect. They are characterized by an expression pattern typical of LEA proteins and seem to protect cells against dehydration by maintaining the structure of proteins and water binding (Close, 1996, 1997; Farrant et al., 2004). During drying after maximum dry matter accumulation, the seed is prepared to survive in adverse weather conditions by reducing the water content inside seed tissues. Such dehydration rates might have harmful effects on cells, tissues and organs, and so protective events must also take place. Recent data indicate that DHNs may play a protective role during dehydration. In this study, we investigated water distribution during seed maturation drying in comparison with spatial and temporal accumulation of DHNs inside Pisum sativum seeds.
1941 15 to 40 d after flowering (DAF). Seeds were removed from pods after harvest and used immediately in order to determine mean seed fresh and dry mass (mg) and seed water content expressed on a dry mass basis (g H2O g 1DM). Freshly harvested seeds collected from 30 to 45 DAF were used for analysis of water distribution by magnetic resonance imaging (MRI). Embryos isolated from seeds were also frozen in liquid nitrogen and used for immunological analysis. Protein extraction Embryos (1 g) were ground with liquid nitrogen, and the powder was homogenized on ice in 2 vols of 20 mM Tris–HCl buffer, pH 7.5, with 5% (v/v) glycerol, 10 mM bmercaptoethanol and 35 mL protease inhibitor cocktail (Sigma) for 1 h. After that time, the homogenates were centrifuged at 13,000g for 20 min. The supernatants were collected and heated at 80 1C for 15 min. After cooling on ice, the probes were centrifuged at 13,000g for 20 min, and the supernatants were taken for further analysis. Protein measurement was performed according to Bradford (1976), using BSA as standard. Electrophoresis and immunodetection of dehydrins Samples of extracts were electrophoresed in 15% polyacrylamide SDS slab gels using a Mini-protean III cell (Bio-Rad, UK). In all, 10 mg of total protein was loaded in each well. Electrophoresis-separated proteins were then transferred onto a PVDF membrane (Immobilon P) using a semi-dry transfer system (Sigma-Aldrich, St Louis, MO, USA). The transfer was carried out at 2 mA for each square cm for 60 min. The membrane was blocked in phosphate buffer saline (PBS) with 5% (w/v) skimmed milk (SM) for 1 h. After blocking, the membrane was incubated overnight at 4 1C with primary anti-DHN antibody (Stressgen Bioreagents Corp., Victoria, Canada) in PBS with 5% SM and 0.05% Tween 20. After three consecutive washes of 5 min each in PBS, the membrane was incubated with biotinylated secondary anti-rabbit antibody (Sigma) in PBS with 5% SM and 0.05% Tween 20 for 2 h. After three washes in PBS, the membrane was incubated for 15 min with streptavidin conjugated with alkaline phosphatase in PBS followed by three washes with PBS. The visualization of DHN protein was carried out with BCIP/NBT (Sigma Fast). Independent protein extractions and immunoblots were performed in triplicate. Tissue printing
Material and methods Plant material Seeds of pea (P. satium L., cv. Piast) were sown in the experimental fields of the Agricultural University of Poznan ´ at Złotniki (near Poznan ´, Poland). Irrigation, fertilization and crop protection were designed to ensure optimal crop growth. Pea pods were hand collected from
Transverse sections of pea cotyledons and embryo axes were made in the median or near-median part of the seeds with a razor blade. The freshly cut surface was briefly wiped on Whatman paper to remove excess of liquid. The tissue was then pressed against the PVDF membrane for approximately 5–10 s, and the membrane was dried at room temperature. The tissue-printed membrane was blocked by incubating in 5% (w/v) SM in
ARTICLE IN PRESS 1942 PBS buffer for 1 h and treated for immunodetection of DHNs as described above. 1
H MRI
Experiments reported here were carried out using an Avance DMX 400 MHz spectrometer (Bruker, Germany) equipped with a standard microimaging birdcage probe head. 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 probe head. For each developmental stage, five seeds were analyzed. The angle between the long axis of the seed and the direction of the B0 field was 901. All images of seeds were acquired using a spin echo pulse sequence (Callaghan, 1991) with soft radio frequency (rf) pulses. Echo time TE, the time between a 901 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 different water content 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 optimize the signal/noise ratio of images. The gain used covered the 26 dB range. Images were reconstructed on a 256 256 pixel matrix with a field of view of 15 mm and slice thickness of 500 mm. Three images were acquired in a perpendicular direction for every seed. All imaging experiments were performed at a temperature of 20 1C.
Results Seed development The changes in fresh mass, dry mass and water mass (expressed on a per seed basis) of pea seeds during their development in planta are presented
M. Garnczarska et al. in Figure 1. An increase in fresh mass occurred from 15 to 30 DAF, while water mass increased until 30 DAF and decreased thereafter. The final water mass was about 45 mg per seed at 45 DAF. Dry mass of the seed increased following a sigmoidal curve. The maximum dry matter accumulation occurred at around 35 DAF.
Magnetic resonance imaging MRI was used to measure the spatial distribution of water in maturing pea seeds from 30 to 45 DAF (Figure 2). MR images were obtained in two orthogonal orientations: axial, or transverse, in which the plane is perpendicular to the embryonic axis; and sagittal, which is parallel to the embryonic axis. The images presented in Figure 2 are limited to one 2D median or near-median slice in the sagittal and axial planes, respectively. The slices were taken as a series of sections from a single seed at a given time. In pea seeds collected at 30 DAF (approximately 1.28 g H2O g 1DM), the most intense signal was observed in the seed coat and the outer part of cotyledons. The signal coming from water was also strong in the vascular network inside cotyledons. In pea seeds at 35 DAF, the water content decreased to 0.98 g H2O g 1DM and the outer parts of seed were characterized by lower 1HNMR signals compared to seeds harvested at 30 DAF. Images of water distribution in pea seeds at 40 DAF showed that the embryo axis was more hydrated than cotyledons, and the outer parts of cotyledons were the areas with more intense signals than the inner parts. The vascular network was still well distinguished. At 45 DAF, the water content was very low and the 1H-NMR signal faded away, and traces of water could only be seen in embryo axis.
Accumulation of dehydrins
Figure 1. Changes in dry, fresh and water mass of pea seeds during their development. Means of three replicates of 15 seeds each 7SD.
Accumulation of DHN proteins was investigated using an immunochemical reaction with an antibody against the DHN C-terminal synthetic peptide based on a DHN consensus sequence (K-segment), with an amino-terminal cysteine (C) residue added (Stressgen). Total soluble heat-stable proteins extracted from pea embryos collected from different seed stages were separated by electrophoresis and immunoblotted. The analysis revealed three polypeptides with molecular masses of 29, 30 and 35 kDa (Figure 3). All polypeptides were present in seeds collected at 15 DAF, but the abundance of DHNs proteins with molecular masses of 30 and 35 kDa increased during maturation. The amount of
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds
1943
Figure 2. MR images of maturing pea seeds from 30 to 45 DAF. Pictures show median or near-median slice in the sagittal (the first row) and axial plane (the second row). Numbers indicate days after flowering. Images were chosen among five views and were selected for being representative of all of the views.
Figure 3. Dehydrins in maturing pea embryos from 30 to 45 DAF. Heat-stable soluble proteins were extracted and 10 mg of protein per lane was loaded on the gel. Dehydrins were detected by western blotting with antiserum raised against the dehydrin consensus polypeptide comprised of residues (C)TGEKKGIMDKIKEKLPGQH (Stressgen). Independent protein extractions and immunoblots were performed in triplicate and yielded identical results.
29 kDa polypeptide decreased slightly during maturation drying. To control for nonspecific reaction of antibodies, blots not incubated with a primary antibody or without a secondary antibody were made and showed no signal. The control reactions without primary and secondary antibodies and streptavidin conjugated with alkaline phosphatase eliminated the possibility of biotinylated proteins having been observed.
Immunolocalization of dehydrins Localization of DHNs in seeds was determined by tissue printing on a PVDF membrane. Tissue
Figure 4. (A) Protovascular localization of dehydrin protein in axial sections performed through pea cotyledons and embryo axis from seed collected at 30 DAF. The experiment was carried our three times with similar results. (B) 1H-NMR signal in axial section of pea seed collected at 30 DAF. axis, embryo axis; cot, cotyledons.
printings were analyzed using the anti-DHN serum. Figure 4 presents tissue prints of seeds collected at 30 DAF. Similar results were obtained with seeds collected at 35 DAF (data not shown). Anti-DHN serum showed strong recognition associated with protovascular tissue, both in the embryo axis and cotyledons. It was not possible to obtain impressions of younger embryos, because the softness and high water content of these tissues precluded clear impressions. It was also too difficult to obtain impressions of dry mature seeds by pressing onto the PVDF membranes.
ARTICLE IN PRESS 1944
Discussion The development of orthodox seeds is terminated by maturation and desiccation, which result in gradual reduction in metabolism as water is lost from seed tissues and the embryo passes into a metabolically inactive or quiescent state (Kermode and Finch-Savage, 2002). Water mass declined during pea seed filling as the dry mass of the seed accumulated (Figure 1). This change in water content was due to water volume being displaced from the expanded cells by the water deposition of reserves within the storage tissues. During desiccation, dry mass accumulation slowed, and fresh mass declined markedly and was accompanied by a rapid decline in water mass (Figure 1). During this phase, there was a net loss of water from the seed. MRI visualized the process of water loss in maturing pea seeds. MR images showed local inhomogeneities of water distribution inside pea seeds (Figure 2). In seeds harvested at 30, 35 and 40 DAF, the intensity of the signal coming from water declined from the inner part of cotyledons tissues. The inhomogeneity of water distribution in pea seeds may be correlated with the gradient of storage substances accumulation within cotyledons. Most seeds lose water as reserves are deposited primarily within storing tissues, displacing water from the cells. In pea seeds, starch can comprise around 50% of the dry mass (Go ´recki, 2001). Starch accumulation begins in parenchyma cells at the inner adaxial region of the cotyledons and spreads to the abaxial region (Borisjuk et al., 1995, 2003). The pattern of water loss may reflect the pattern of starch deposition in cotyledons. MR images of lupine seeds showed that, in contrast to pea seeds, the decline of the 1H-NMR signal intensities was more rapid in the outer parts of the seed than in the central part (Garnczarska et al., 2007) and water appeared to evaporate from the seed surface. The differences in the route and mechanism of water loss between pea and lupine may be associated with differences in seed reserves composition, since proteins constitute up to 45% of the DM of lupine seed and starch represents only up to 2% of the DM of the seed (data not shown). However, this may not be only because of differences in water-absorbing capacity (i.e. hydroscopic properties) of pea and lupine, since barriers to water movement might also influence the drying rate of seeds. At the end of the seed filling stage, the abrupt reduction in pea seed water mass and MR image intensity may also correspond to an interruption of water supply to the pod as well as the senescence of the pod. During drying after maximum dry matter accumulation, the differ-
M. Garnczarska et al. ences in the intensity of the signals between cotyledons and the embryonic axis became apparent. In seeds harvested at 40 DAF, the embryonic axis appeared to be more hydrated than cotyledons in contrast to earlier stages, and even in dry mature seeds traces of water could be seen in the axis. One explanation is that dry matter accumulated in reserve-storing tissues does not have the same hydroscopic properties as materials in the embryonic axes. It can be a part of the protective mechanism since, according to Vertucci and Leopold (1984), tissues could be somewhat protected from imbibitional injury by elevating the initial moisture level. The 1HNMR 2D micro-imaging of low-hydrated tobacco seeds also showed that there were localized tissue areas in the covering layers with higher proton mobility (Leubner-Metzger, 2005). This finding indicates the presence of free water in air-dry seeds. Proton NMR indicated retention of mobile water during dehydration in the desiccation-tolerant grass Eragrostic nindensis (Balsamo et al., 2005) as part of the strategy for surviving water loss in vegetative tissues. Drying after maximum dry matter accumulation is either coincident with or subsequent to the acquisition of desiccation tolerance. It has been demonstrated that DHNs, a group-2 LEA class of proteins, accumulate under conditions of water deficit (Close, 1997; Farrant et al., 2004). They are expected to play a protective role during dehydration; however, functional evidence is scare. Immunological analyses of DHN accumulation were performed in pea embryos isolated from seeds from 30 to 45 DAF. Western blots showed that the temporal pattern of accumulation of two DHNs with molecular masses of 30 and 35 kDa correlated well with seed maturation (Figure 3). This suggests the involvement of these proteins in protective reactions promoting maintenance of embryo structures under conditions of water deficit (Allagulova et al., 2003). The amino acid composition of DHNs with high content of charged and polar residues may promote their specific protective functions under conditions of cell dehydration (Allagulova et al., 2003; Rorat, 2006). Immunolocalization by tissue printing showed DHN proteins to be associated with protovascular tissues of both the embryo axis and cotyledons (Figure 4). This pattern of DHNs localization corresponds well with water distribution inside pea seeds. The intense 1H-NMR signal appeared in seeds collected at 35 DAF, and referred to protovascular bundles. As shown by Bauby et al. (2007), during Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell division, giving rise to vascular precursors cells
ARTICLE IN PRESS Water and dehydrin distribution in maturing pea seeds (protophloem and protoxylem precursors) in developing organs. DHNs present in the embryonic axis may increase its water-absorption capacity. The localization of DHN protein in vascular tissues was also observed in maturing lupine embryos (Garnczarska et al., 2007) and in barley under cold acclimation (Bravo et al., 1999). DHNs are highly hydrophilic proteins, which might explain the more intense signal coming from water in MR images in the vascular bundle region inside seeds. Vascular localization of DHNs might promote water influx into the vascular bundles. DHNs may function as water attractants during the transport of water and seed reserves to sink tissue (Rorat, 2006).
Conclusions The loss of the water signal in maturing pea seeds proceeds gradually from the inner to the outer part of cotyledons and is likely orientated in the same direction as starch accumulation. The pattern of DHN localization reflects the pattern of water distribution in the protovascular bundles region of maturing pea embryos, suggesting the involvement of these proteins in promoting water influx into the vascular bundles.
Acknowledgment This work was supported by the State Committee for Scientific Research (KBN), Grant 2 P06R 085 26.
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