JXB Advance Access originally published online on May 24, 2007
Journal of Experimental Botany 2007 58(8):2261-2268; doi:10.1093/jxb/erm101
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RESEARCH PAPER |
Localization of myo-inositol phosphate synthase (GmMIPS-1) during the early stages of soybean seed development
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA
To whom correspondence should be addressed. E-mail: egrabau{at}vt.edu
Received 2 March 2007; Revised 6 April 2007 Accepted 17 April 2007
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Abstract |
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As a precursor to a large variety of compounds, myo-inositol is a central molecule required for cell metabolism and plant growth. The de novo synthesis of myo-inositol requires the activity of the enzyme D-myo-inositol-3-phosphate synthase (MIPS). MIPS cDNAs encoding one or more isoforms have been cloned from a number of species, nevertheless, little is known about the regulation of MIPS expression in developing seed. Seed-specific expression of a soybean isoform (GmMIPS-1) has been demonstrated, but tissue-specific localization during embryo development has not been reported. Using immunolocalization techniques, a specialized area of GmMIPS-1 expression was identified in the outer integumentary layer during early soybean seed development. In addition, localization data provided evidence that MIPS was associated with oxalate crystal idioblasts.
Key words: Calcium oxalate, development, Glycine max, crystal idioblasts, MIPS, myo-inositol, myo-inositol phosphate synthase, soybean seed
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The biosynthesis of myo-inositol is critical to the production of a functionally diverse group of compounds. Inositol, a six-carbon cyclitol, is produced through the conversion of D-glucose-6-phosphate to D-myo-inositol-3-phosphate by the isomerase D-myo-inositol-3-phosphate synthase (MIPS, EC 5.5.1.4 [EC] ). Subsequent phosphatase activity produces free inositol, the precursor for many inositol-derived compounds found in plants (Loewus and Murthy, 2000). Compounds derived from myo-inositol participate in activities such as signal transduction (phosphatidylinositols), stress adjustment (pinitol and ononitol), and seed storage (raffinose, phytic acid).
Several plants possess multiple isoforms of the D-myo-inositol-3-phosphate synthase (MIPS) enzyme, suggesting that each gene copy may be differentially controlled and expressed. Multiple isoforms of MIPS were reported for Zea mays (Larson and Raboy, 1999), Phaseolus vulgaris (Johnson and Wang, 1996), and Arabidopsis thaliana (Johnson, 1994; Johnson and Burk, 1995). Differential expression was observed in P. vulgaris with a smaller MIPS protein expressed during the globular stage of embryogenesis and a larger MIPS protein expressed during the cotyledonary stage of embryogenesis (Johnson and Wang, 1996). Soybean contains four MIPS isoforms and one of the MIPS cDNAs (designated GmMIPS-1) was shown to express mainly in developing seeds (Hegeman et al., 2001; Chappell et al., 2006).
DNA sequences for MIPS have been cloned from a variety of organisms including yeast (Johnson and Henry, 1989), Citrus paradise (Abu-Abied and Holland, 1994), Glycine max (Hegeman et al., 2001), Passiflora edulis (Abreu and Aragão, 2007), and Oryza sativa (Mizobuchi-Fukuoka et al., 1996). To date, however, only the localization of the RINO1 transcript of rice has been characterized in developing seed (Yoshida et al., 1999). In situ hybridization showed that the RINO1 transcript was first detected in the apical cells of the embryo, but as the seed developed, the transcript appeared in the scutellum and aleurone layer (Yoshida et al., 1999). Localization data revealed the dynamics of gene expression and provided a more detailed analysis of expression.
In order to characterize GmMIPS-1 further, immunological techniques were used to localize protein expression in developing soybean seeds. Sections of developing soybean seeds from four stages of development were used to characterize early GmMIPS-1 expression. A unique pattern of abundant GmMIPS-1 expression was observed in the integumentary layers of the developing seeds. In addition, expression was found in crystal idioblasts, indicating that GmMIPS-1 was associated with the formation of calcium oxalate crystals.
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Materials and methods |
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Plant growth conditions and tissue sampling
Soybean plants (Glycine max cv. Jack) were grown in the greenhouse with supplementary light from high pressure sodium vapour lights under a 16/8 h light/dark photoperiod. Day/night temperatures were a constant 28 °C. Plants were fertilized after the emergence of the fourth trifoliolate leaf with Micracle-Gro (Scotts, Marysville, OH, USA) at 0.25x the manufacturer's recommended rate.
Seed pods were collected from soybean plants at different developmental stages over a period of several weeks after flowering. Developing seeds smaller than 5.0 mm were excised from the pod and sorted according to seed size before placement into a PIPES fixing buffer containing 50 mM 1,4-piperazinebis(ethanesulphonic acid), 5 mM MgSO4, 5 mM EDTA, 1 mM spermidine, 0.1% Tween 20, and 4% formaldehyde (pH 7.0). Tissues were fixed overnight at 4 °C, and then washed three times with several volumes of the PIPES buffer without formaldehyde.
Tissue embedding and sectioning
Fixed and washed tissues were dehydrated through an ethanol series (50, 70, 95%) and infiltrated with either ImmunoBed (<3 mm seeds) or OsteoBed (>3 mm seeds) embedding medium and polymerized according to the manufacturer's directions (Polysciences, Inc., Warrington, PA, USA). 5 µm sections were made from embedded material with a Reichert Jung 2040 microtome (Leica Microsystems, Bannockburn, IL, USA) and placed onto Fisher Biotech ‘probe-on’ microscope slides (Fisher Scientific, Pittsburgh, PA, USA). For the Osteo-Bed embedded tissues, the polymer-resin was removed by placing the slide into Osteo-Bed solvent.
MIPS immunolocalization
Slides with tissue sections were rinsed in 1x phosphate buffer saline (PBS) solution (140 mM NaCl, 2 mM KCl, 10 mM Na2HPO4, 1 mM KH2PO4, pH 7.0) containing 0.1% bovine serum albumin (BSA) for 15 min and blocked for 1 h in PBS/BSA containing 10% goat serum (Sigma, St Louis, MO, USA). Sections were washed with PBS/BSA for 10 min and incubated for 16 h with anti-GmMIPS-1 polyclonal primary antibody (Styer, 2000) diluted 1:50 in PBS/BSA. The primary antibody was removed by three washes with PBS/BSA for 10 min each, followed by incubation for 4 h with a rhodamine-conjugated goat anti-rabbit F(ab')2 specific antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) diluted 1:100 in PBS/BSA. Sections were washed three times with PBS/BSA for 10 min and cover slips were mounted with 90% glycerol containing 1 µg ml–1 4',6-diamidino-2-phenylindole (DAPI).
Sections were examined using a Zeiss Axioskop microscope (Carl Zeiss Inc., Thornwood, NY, USA) equipped with differential interference contrast (DIC) and fluorescence filter sets for DAPI (Ex 350 nm/Em 460 nm) and TRITC fluorescence (Ex 540 nm/Em 605 nm; Chroma Technology Corp., Rockingham, VT, USA). DIC, DAPI, and TRITC images were captured using a Spot 2 digital camera and software (Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Colour composites of either three (DAPI, DIC, TRITC) or two (DIC, TRITC) images were made by using the screen overlay method in Photoshop 7.0 (Adobe, Mountain View, CA, USA).
Only representative images of expression patterns that were consistently observed in sections of three or more individual seeds from each developmental stage were selected for presentation. Brightfield images of representative sections stained with toluidine blue O and basic fuschin were also captured.
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Results |
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GmMIPS-1 localization during the globular embryo stage
Seeds at the globular embryo stage had an acellular endosperm, a large central vacuole, and a globular embryo; all were defining features of this stage of development (Dute and Peterson, 1992; Fig. 1A). No background localization was observed in control sections incubated with only the secondary antibody (Fig. 1B). In cells of the integumentary layers, GmMIPS-1 was localized to vesicular structures that were adjacent to nuclei (Fig. 1C, E). In the developing cellular endosperm, GmMIPS-1 localization was punctate, but expression in the nucellar tissue appeared to be dispersed throughout the cell (Fig. 1D). A significant GmMIPS-1 localization signal was observed at opposite ends of the seed, adjacent to the globular embryo, and at the chalazal end (Fig. 1D, E). GmMIPS-1 was expressed at the interface between the embryo and the embryo sac and corresponded to a light-blue staining area near the micropyle observed in the toluidine blue O- and basic fuschin-stained sections (Fig. 1A, D). At the chalazal end of the seed, GmMIPS-1 was localized to the endosperm haustorium in three plastid-like structures (Fig. 1C).
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GmMIPS-1 localization during the globular-heart stage
The inner and outer integumentary layers were easily discernible in brightfield images of the globular–heart-staged seed (Fig. 2A). At this stage, cells of the outer integument, adjacent to the micropyle, appeared larger and more vacuolated than the cells toward the chalazal end of the seed. A punctate pattern of localization was observed in most cells of the outer integumentary layer, but a particularly strong localization signal was observed in a group of cells located in the outer integumentary layer region near the micropyle (Fig. 2B, C). GmMIPS-1 localization in these cells appeared dispersed throughout the cell and the expressing cells abruptly terminated at the micropylar pore (Fig. 2C, E). GmMIPS-1 expression also appeared evenly dispersed in cells of the inner integument layer adjacent to the integumentary tapetum near the micropylar end of the seed (Fig. 2C). Expression in this region, however, was much lower compared with the group of cells adjacent to the micropyle. GmMIPS-1 signal was detected in the endosperm and at the chalazal end of the seed in the cellular haustorium, but the localization signal was more intense at the interface between the integumentary tapetum and the cellular haustorium (Fig. 2D). Auto-fluorescing particles were present at this stage, but were easily discernible from the fluorescence pattern of GmMIPS-1 (Fig. 2F).
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GmMIPS-1 localization during the early cotyledonary stage
At the early cotyledonary stage, the endosperm was well-developed and the embryo had not yet rotated (Fig. 3A). Cells of the seed coat epidermal and hypodermal layers were well differentiated and the inner and outer integumentary layers, including the integumentary tapetum, were well developed. GmMIPS-1 expression was observed between the embryo and the endosperm at the micropylar end of the seed, as well as in adjacent cells (Fig. 3B). GmMIPS-1 localization signal was observed in the epidermal cells where expression was punctate (Fig. 3C). By contrast, in the differentiating hypodermal layer, the localization signal appeared more diffuse (Fig. 3C). Most obvious was the intense GmMIPS-1 signal present in the micropylar region, but appeared in cells slightly further from the micropylar pore (Fig. 3C, D). No signal was detected in control sections (Fig. 3E).
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GmMIPS-1 localization during the cotyledonary stage
At the mid-cotyledonary stage, the embryo had rotated, the cotyledons had expanded into the endosperm, and the primary leaves were initiated (Fig. 4A). Control sections did not reveal any background GmMIPS-1 signal (Fig. 4B). Unlike the previous two stages of seed development, a localization signal for GmMIPS-1 was not detected in the outer integument near the micropyle. Instead, a lower intensity GmMIPS-1 localization signal was detected throughout the seed, including in the radicle of the embryo. GmMIPS-1 expression in the cotyledons appeared to be evenly distributed throughout cotyledonary cells (Fig. 4C). A punctate pattern of GmMIPS-1 expression was observed in the radicle of the embryo (Fig. 4E), but was not detected in the apical regions of the embryo. In maternal tissues, a diffuse signal was observed in cells of the outer integumentary layer, but was much further from the micropyle (Fig. 4D).
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GmMIPS-1, crystal idioblasts and the radicle
At the mid-to-late cotyledonary stages, GmMIPS-1 was detected in crystal idioblasts and in cells of the radicle (Fig. 5). Low levels of GmMIPS-1 signal was detected in crystal idioblasts where calcium oxalate crystals of various developmental stages were located (Fig. 5A). At a late stage of crystal development, GmMIPS-1 signal was detected in the narrow channel between the oxalate crystal wall and the cell wall (Fig. 5C). In the radicle of the embryo, GmMIPS-1 expression was punctate and associated with vesicular structures (Fig. 5B, D). No cross-reacting MIPS protein was detected in the apical region of the embryo (data not shown).
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Discussion |
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Using immunological techniques, the expression of the soybean GmMIPS-1 was characterized during early seed development. Even though a polyclonal antibody was used in the study, artefactual labelling was unlikely because molecular evidence has shown that only one MIPS isoform, GmMIPS-1, was highly expressed in developing seeds (Hegeman et al., 2001; Chappell et al., 2006). At these early stages of seed development, GmMIPS-1 was expressed in maternal tissues and then in the developing embryo.
MIPS expression was localized to vesicular structures during the globular embryo stage of seed development and to the micropylar and chalazal ends of the seed (Fig. 1). A study involving the 14C labelling of photoassimilates showed that assimilates concentrated at the micropylar and chalazal ends of the seed during the zygotic and globular stages of embryo development (Chamberlin et al., 1993). It was determined that assimilates were passed through the chalazal process and the suspensor to the endosperm and embryo (Chamberlin et al., 1993). In this study, MIPS was also observed to localize in the same regions of the seed as did the labelled assimilates. Together, these data indicate that specific regions of the developing seed supply a large amount of the required myo-inositol for embryo and endosperm development.
A particularly distinct GmMIPS-1 localization pattern occurred on one side of the micropyle, in a group of cells located in the outer integument during the heart and cotyledonary stages of embryo development (Figs 2, 3). Generally, the outer integument has been viewed as a more or less homogeneous mass of cells. The GmMIPS-1 localization pattern illustrated here offers a more defined region within the outer integumentary layer that can be described as a micropylar complex. In cells of the micropylar complex, GmMIPS-1 expression was distributed throughout, signifying cytoplasmic localization (Fig. 3). Given the intense fluorescence of the MIPS signal, one of the major functions of these cells was presumably to produce myo-inositol. Whether the myo-inositol was retained or exported from the cell is unknown, but its proximity to the micropyle suggests that myo-inositol was exported to the embryo via the suspensor (Yeung, 1980; Nagl, 1990). In addition, a study using ‘seed coat cups’ of soybean seeds, determined that myo-inositol was produced in the seed coat (maternal tissues) and transported to the embryo (Gomes et al., 2005).
A number of crystal idioblasts in the embryo contained either developing or well-defined calcium oxalate crystals (Fig. 5A, B). Calcium oxalate crystals are commonly observed in plants (Arnott and Webb, 1983) and are particularly abundant in developing seeds of soybean (Ilarslan et al., 1997; Horner et al., 2005). Calcium oxalate crystals modulate physiological calcium levels within plant tissues (Webb, 1999; Franceschi and Nakata, 2005) and, depending on the calcium requirement of the developing soybean seed, the number and distribution of calcium oxalate crystals changes as the seed develops and matures (Ilarslan et al., 2001). In the present study, a wall extending from the crystal vacuole to the cell wall was clearly visible in sections of the crystal idioblasts (Fig. 5C). A similar structure, described as a sheath in Phyllanthus, functioned as a conduit between the cell and the crystal forming vacuole (Grimson and Arnott, 1983). Since crystals could either be disappearing or developing, the direction of transport in this study was unknown. Nonetheless, GmMIPS-1 was localized within the wall surrounding the crystal and, therefore, must be supplying myo-inositol for either the crystal development or the wall surrounding the crystal. Both the biosynthesis of calcium oxalate and the wall surrounding the crystal require myo-inositol as a precursor. The production of the calcium oxalate crystal via ascorbic acid requires myo-inositol (Lorence et al., 2004) and the wall surrounding the crystal is also composed of inositol derivatives such as cellulose, pectin, and suberin (Scott, 1941; Wattendorff, 1978).
A limited amount of information about GmMIPS-1 expression in developing seed was known from previous analyses of the GmMIPS-1 family (Hegeman et al., 2001; Chappell et al., 2006). In developing rice embryos, the analogous RINO1 transcript was first detected at the apex of embryos and then in the scutellum and aleurone layer (Yoshida et al., 1999). In this study, unlike in rice, the GmMIPS-1 expression was first detected in maternal tissues and then in the embryo. Even within the embryo, expression was first observed in the radicle rather than the apical regions. GmMIPS-1 expression in the maternal tissue is likely to be the primary inositol supply for the early embryo. Using RNAi-silencing methods, GmMIPS-1 expression was silenced and resulted in aborted seed, but partial silencing produced viable seed (Nunes et al., 2006). The localization and silencing data illustrate that GmMIPS-1 expression is vitally important for the initial developmental stages of the embryo.
Using immunological techniques, the present study revealed a unique GmMIPS-1 micropylar complex and the association of GmMIPS-1 with calcium oxalate crystal idioblasts. In addition to characterizing GmMIPS-1 in embryos at later developmental stages, three additional members of the soybean MIPS family, each with a unique expression pattern during plant development, need to be characterized to fully understand myo-inositol metabolism.
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Acknowledgements |
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The authors wish to thank Dr JJ Finer for critical review of the manuscript. The research was supported by a grant from the USDA National Research Initiative Competitive Grants Program.
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Footnotes |
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* Present address: Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, Ohio State University, Wooster, Ohio 44691, USA.
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