Botanical Studies (2010) 51: 183-194.
CYTOLOGY
Tissue- and cellular localization of soybean (Glycine max L.) seed maturation protein transcripts
Ming-Der SHIHl, Mei-Hua KAO2, Shu-Chin LIN2, Tzu-Fu SHU1, Kim-Long HSIEH1, Mei-Chu CHUNG1, Tze-Hua TSOU1, Yue-Ie C. HSING1, and Jaw-Shu HSIEH2,*
1Institute of Plant and Microbial Biology, Academia Sinica, Taipei 11529, Taiwan
2Department of Agronomy, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan (Received November 9, 2009; Accepted January 20, 2010)
ABSTRACT. Late embryogenesis abundant (LEA) genes, with abundant expression in seeds during the late developmental stages, are associated with water loss. In the present work, we characterized the spatial ex­pression patterns of 8 soybean GmPM genes, including 7 hydrophilic LEAs and 1 maturation protein gene, in fresh or pod-dried seeds at 35 days after flowering (DAF) by in situ hybridization analysis. The transcripts of hydrophilic LEA genes were detectable in vascular tissues of the cotyledon and embryo axis (epicotyl and radicle) in fresh seeds 35 DAF. After pod-drying, the expression of these genes was greatly increased in similar tissues and was induced in other seed tissues. A maturation protein gene, GmPM5, was highly expressed in mesophyll tissues but not in vascular tissues of fresh or pod-dried seeds. Hence, LEA genes could be be highly regulated in the conductive system with changes in water content. As well, the mRNA distribu­tion of GmPM5 and GmPM8/10, 2 messengers with endoplasmic reticulum signal peptides in the deduced proteins, significantly differed. The transcripts of these genes are therefore targeted towards different endo­plasmic reticulum systems, which suggests that individual endoplasmic reticulum-targeting LEA genes may be involved in diverse translocation and subsequent protein transport systems.
Keywords: Endoplasmic reticulum; GmPM; In situ hybridization; Late embryogenesis abundant; Soybean; Spatial expression patterns; Vascular bundle.
INTRODUCTION
Water loss or desiccation could be an important part of the maturation process in orthodox seeds (Rosenberg and Rinne, 1986). During this process, gene expression and protein-synthesized profiles are markedly changed, which is usually associated with achieving desiccation tolerance, increased abscisic acid (ABA) content, or the capability of seed germination (Goldberg et al., 1989; Hughes and Galau, 1989; Skriver and Mundy, 1990). LEA genes were first identified from embryos of mature wheat (Triticum aestivum) (Cuming et al., 1984) and cotton (Gossypium hirsutum) (Galau et al., 1986). They are highly expressed during the late seed developmental stage or are induced by exogenesis ABA or environmental stress in vegetative tissues (see reviews in Dure et al., 1989; Ingram and Bartels, 1996; Cuming, 1999). LEA genes are not plant specific. Several LEA genes have been described from bacteria, fungi, or anhydrobiotic animals; examples are Bacillus subtilis (Mueller et al., 1992), cyanobacteria (Close and Lammers, 1993), Tuber borchii (Abba' et al., 2006), nematodes (Tunnacliffe and Wise, 2007), rotifers (Adineta ricciae) (Pouchkina-Stantcheva et al., 2007),
brine shrimp (Artemia franciscana) (Hand et al., 2006; Wang et al., 2007), and Polypedilum vanderplanki (Ki-kawada et al., 2006). This wide distribution suggests the importance of LEA genes under desiccation conditions.
The deduced LEA protein sequences are classified into at least 5 groups according to their conserved motifs or sequence similarity (Shih et al., 2008). Group I to IV LEA proteins are highly hydrophilic, and the remain­der are hydrophobic. Hydrophilic LEA proteins lack defined secondary structures in solution status and belong to the members of "natively unfolded proteins" (see review in Shih et al., 2008 and references). However, the detailed 3-D structures of At1g01470.1, the hydrophobic Arabidopsis D-95 type LEA protein, were determined as ap-fold conformations by NMR spectroscopy (Singh et al., 2005). Except for group I to V LEA genes, some genes which encoded proteins lack the motifs or protein characteristics defined in other LEA proteins also express in seeds during late embryogenesis and disappear after the early hours of germination (Shih et al., 2008). Unlike typical LEA proteins, they may be carrier proteins, enzymes, fiber proteins, or plasma membrane proteins (e.g. Shen et al., 1993; Koike et al., 1997; Hsmg et al., 1998; Breton et al., 2003). The overexpression of LEA genes was reported to improve stress tolerance of transgenic plants or recombinant cell lines. For example, transgenic
*Corresponding author: E-mail: jawar@ntu.edu.tw; Tel: +886-2-33664756; Fax: +886-2-23620879.
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cereals harboring barley HAV1 or wheat PMA80 and PMA1959 showed tolerance to drought or salinity stresses (Xu et al., 1996; Cheng et al., 2002). Several group II LEA (dehydrin) genes introduced into target plants also increased resistance to chilling, freezing, free radicals, or salinity (Hara et al., 2003; Houde et al., 2004; Puhakainen et al., 2004; Figueras et al., 2004). Additionally, a series of experiments with recombinant cell lines showed increased stress tolerance with the accumulation of hydrophilic LEA proteins (Imai et al., 1996; Swire-Clark and Marcotte, 1999; Zhang et al., 2000; Chakrabortee et al., 2007).
Previous work suggested that changes in seed constituents and levels of several newly synthesized proteins were similar between natural and precocious maturation (Rosenberg and Rinne, 1987). Precocious maturation was used as a model system to investigate the control of the physiological and biochemical events occur­ring during seed maturation (Rosenberg and Rinne1988; Hsing et al., 1990). A cDNA library was prepared from 4-day pod-dried (PD) soybean seeds at 35 days after flow­ering (DAF), followed by differential screening (Hsing et al., 1990; Hsing and Wu, 1992). Forty-one cDNA clones designated Glycine max physiological mature (GmPM) were sequenced, and 22 of them belonged to typical LEA genes. We already characterized some of these genes by studying the expression profiles under hormone or osmotic treatments, genetics, or their structural biology (Hsing and Wu, 1992; Hsing et al., 1995; Hsing et al., 1998; Lee et al., 2000; Shih et al., 2004; Tsai et al., 2008).
However, information on spatial expression patterns was still missing. Here, we present the results of cellular localization analysis of these transcripts, including GmPM11 (group I), GmPM6/7 and GmPM12 (group II), GmPM2, GmPM8/10, and GmPM30 (group III), GmPM1 (group IV), and GmPM5 (maturation protein gene). The transcripts of most of the tested hydrophilic LEA genes accumulated in vascular tissues of the cotyledon and embryo axis (epicotyl and radicle). As well, the endoplas-mic reticulum (ER)-targeting LEA genes, GmPM5 and GmPM8/10, showed different mRNA distribution in coty-ledon mesophyll cells.
MATERIALS AND METHODS
Plant materials
Cultivated soybean (cv. Shih-shih) seeds were kindly provided by Kaohsiung Agricultural Experimental Station, Pintoung, Taiwan. Plants were grown in the field under a normal day-length period. Pods at 35 DAF that were fresh or intact pods precociously matured by air-drying (PD) for 4 days were harvested as described previously (Hsing et al., 1990; Hsing and Wu, 1992).
In situ hybridization
In situ hybridization of mRNA was performed as de­scribed previously (Hsing et al., 1998). Soybean seeds at 35 DAF with or without 4-day PD treatment were
harvested and immediately fixed with 4% formaldehyde in 10 mM phosphate buffered saline (pH 7.0) for 16 h at 4°C. The plant materials were washed with 0.85% NaCl solution, dehydrated in a graded ethanol series, and em­bedded in paraffin. Sections (6 to 8 fim thick) were cut and mounted on Vactabond-treated slides. The slides were put in a 42°C oven for 16 h and then stored at 4°C for further use. The linearized GmPM1, 2, 5, 6/7, 8/10, 11, 12, and 30 plasmids were used as templates for the digoxigenin-labeled antisense or sense strands transcribed by T7 or T3 RNA polymerase from their corresponsive clones. Labeling with digoxigenin followed the manufacturer's instructions (Roche, Germany). Probes were hydrolysed to an average size of 150 bp at 60°C before use. Paraffin was removed from the tissue sections by incubation in a series of Histoclear solution, 100% ethanol, and ethanol with 0.85% (w/v) NaCl. Slides were treated with pronase for 4 h at 37°C, then 2 min in 0.2% glycine, 10 min in 4% formaldehyde, and 30 sec each in a series of ethanol solutions. Hybridization of the probes to the slides was performed at 50°C for 8 to 24 h, depending on the clone or tissue. After hybridization, slides were washed several times with NTE solution (500 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 10 mM dithiothreitol) at 37°C. Digoxigenin-labelled RNA probes were then detected by use of an antidigoxigenin alkaline-phosphate conjugate (Roche, Germany). Slides were dehydrated before drying at 50°C and observed under a microscope.
Tissue printing
The nitrocellulose membrane was soaked in 0.2 M CaCl2 solution for 30 min, and dried on paper towels. Fresh cut tissue was washed in distilled water for 5 sec, and blotted onto the membrane for 5, 15, 30, 60, 90, and 120 sec. The tissue print was blocked for 30 min in phosphate-buffered salme/Tween 20 (PBST) (0.1% [w/v] Tween 20, 150 mM NaCl, 50 mM Na2HPO4, and 50 mM NaH2P〇4) with 5% (w/v) defatted dry milk. The sera against GmPM5 protein (1:50,000 dilution) were used for primary labeling. The signals were then amplified by goat anti-rabbit AP-conjugate antibody (Sigma, USA) (1:5,000 dilution). After a PBST/PBS rinse, the color sub­strates (Roche, Germany) were used for GmPM5 protein detection.
RESULTS AND DISCUSSION
The localization of Group I LEA messages
Group I LEA genes encode proteins that contain a con­served 20-mer motif that can be unique or repeated 2 to 4 times in tandem (Espelund et al., 1992). Previous work had divided LEA I proteins into 2 subgroups according to the repeat number of the 20-mer motif. Em1-type proteins contain 2 to 4 repeats, and Em6-type proteins only one (Delseny et al., 2001). One soybean LEA I transcript, GmPM11 (accession no. AF004805), which encodes an Em6-type protein, was used in the current study. The
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predicted molecular mass is 9 kDa, at pI 7.5. The spatial expression patterns of GmPM11 were analyzed in fresh or 4-day PD seeds at 35 DAF (Figure 1). In fresh seeds, GmPM11 transcripts highly accumulate in vascular tissues of radicle but are barely detected in cotyledon vascular strands and radicle cortex (Figure 1B to E). In 4-day PD seeds, GmPM11 mRNAs accumulate in all seed tissue, including the cotyledon and embryo axis (Figure 1F to J). Strong signals are observed in vascular tissues and in the adaxial epidermis cells of cotyledon.
Several Em6-type LEA genes had been studied by in situ hybridization. Carrot Emb-1 mRNA was preferentially detected in the procambial meristematic region of somatic or zygotic embryos from the globular to mature torpedo stages and was greatly accumulated in cotyledons at a later stage (Wurtele et al. , 1 993 ). Sunflower ds11 transcripts appeared to accumulate to high levels in early embryogenesis. In mature seeds, the distribution of ds11 mRNA showed marked localization in vascular tissues of the procambium or cotyledon (Prieto-Dapena et al., 1999). Arabidopsis AtEm6 transcripts were detected throughout the entire embryo in provascular tissues and shoot apical meristem (Vicient et al., 2000). Therefore, the in situ hybridization studies of other Em6-type LEA I genes showed a similar signal distribution in seeds or em­bryos similar to what we found. These results suggested that the messages of Em6-type LEA I genes appear from the early to middle stages of embryogenesis and reach a plateau at the late developmental stage, with preferential accumulation in vascular tissues of the procambium of the cotyledon or embryo axis.
Localization of Group II LEA messages
Group II LEA proteins have 3 specific, characteristic sequence "modules" (i.e., K-domain, Y-domain, and S-segment) that define this class of protein (Close, 1997). In the present study, we used these soybean dehydrin genes (i.e., GmPM6/7 and GmPM12) to illustrate their spatial patterns. GmPM6/7 (accession nos. M9401 2 and L00921) share 96% cDNA sequence identity. The deduced protein sequences of GmPM6/7 belong to Y2K-type dehydrins, with molecular mass of 31/32 kDa and pI 6.6/6.5, respectively. The mRNA of GmPM6/7 (the
2 transcripts cannot be distinguished because of high identity) accumulates in vascular strands, mesophyll around vascular tissues, and adaxial epidermis of cotyledon, epicotyl, as well as cortex, pith, and phloem cells of radicle in fresh seed (Figure 2B to F). By contrast, no or few GmPM6/7 transcript is detected in the abaxial epidermis of cotyledon (Figure 2B and C) and radicle pericycle (Figure 2D). The mRNA expression of GmPM6/7 mRNA is greatly increased in the same regions in PD seeds as in fresh seeds (Figure 2G to L). The mRNA level of GmPM6/7 is increased slightly in radicle pericycle and of cotyledon vascular strands after drying (Figure 2J and K).
The deduced protein sequence of GmPM12 (accession
no. AF004807) shows it to be a Y2SK2-type dehydrin. The molecular mass is 10 kDa at pI 10. In fresh seeds, no or few transcript of GmPM12 in seeds (Figure 3B-F). In PD seeds, the expression of GmPM12 is significantly increased in vascular tissues of the cotyledon and embryo axis (Figure 3H to K).
Figure 1. Localization of soybean GmPM11 transcripts in fresh (panel A to E) or 4-day pod-dried (PD) (panel F to J) soybean seeds. In situ hybridization was carried out with transverse sections of seeds in this and following figures. A sense probe was used in panel A and F and an antisense probe in the remaining panels. Bar=200 (im for panel A, B, F, and G, 60 (im for panel C and H, 40 (im for panel E and J, and 20 (im for panel D, and I. Abbreviations (all the abbreviations is noted at first time): BE, abaxial epidermis; C, cotyledon; Co, cortex; Cp, palisade tissue of cotyledon; DE, adaxial epidermis; P, pith; Ph, phloem; R, radicle; VS, vascular strand; Xy, xylem. Arrowheads indicate cotyledon vascular strands.
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Figure 2. Localization of soybean GmPM6/7 transcripts in fresh (panel A to F) or 4-day PD (panel G to L) soybean seeds. A sense probe was used in panels A and G and an antisense probe in the remaining panels. Bar=200 (im for panel A, B, G, and H, 80 (im for panel C, F, I, and L, 60 (im for panel D and J, 20 (im for panel E and K. Abbreviations: Cs, spongy tissue of cotyledon; E, epicotyl; En, endosperm; Pe, pericycle. Arrowheads indicate cotyledon vascular strands (panel B, C, H, I) or epicotyl procambium (panel F, L).
Several types of dehydrins had been studied by in situ hybridization. The spatial patterns of a carrot Y2SK2-type dehydrin, Ecp40, showed that the transcripts accumulated in embryo and endosperm in dry seeds (Kiyosue et al., 1 993). The mRNA expression of maize (Zea mays) Rab17, an YSK2-type dehydrin, was first detected in leaf primordia and radicle, with little expression in scutellum. During maturation, Rab17 transcripts accumulated in the cortex, protoxylem, and metaxylem cells of radicle and leaf primordia, as well as the procambium and epidmeris cells of the scutellum (Goday et al., 1994). The rice (Oryza sativa) homolog of Rab17, Rab21, showed similar expression patterns, except that Rab21 transcripts were relatively scarce in leaf primordia and the radicle (Miyoshi et al., 1999). These studies indicate that the
mRNA expression of dehydrins is mainly in the embryo axis and vascular bundles. They may provide a protective mechanism primarily for embryos.
Localization of Group III LEA messages
The most significant feature of group III LEA proteins is the tandem-repeating 11-mer motifs. According to the different consensus sequences of 11-mer repeats, LEA III proteins were classified as subgroups D-7, Dc8, GmPM8, and D-29 (Shih et al., 2008). In this paper, we characterize 3 soybean LEA III genes. They are a Dc8 type, GmPM2 (accession no. M80664) (Hsing et al., 1992); two GmPM8 types, GmPM8 and GmPM10 (accession nos. X63565 and U02966) (Hsing et al., 1995); and one D7 type, GmPM30 (accession no. AF117884). The GmPM2 protein contains
Figure 3. Localization of soybean GmPM12 transcripts in fresh (panel A to F) or 4-day PD (panel G to K) soybean seeds. A sense probe was used in panel A and G and an antisense probe in the remaining panels. Bar=200 (im for panel A, B, C, G, and H, 80 (im for panel F and K, 60 (m for panel D and I, and 20 (m for panel E and J. Arrowheads indicate cotyledon vascular strands (panel C, H) or epicotyl procambium (panel F, K).
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Figure 4. Localization of soybean GmPM2 transcripts in fresh (panel A to E) or 4-day PD (panel F to J) soybean seeds. A sense probe was used in panel A and F and an antisense probe in the remaining panels. Bar=200 (im for panel A, B, F, and G, 80 (im for panel E, 60 (m for panel C, H, and J, and 20 (m for panel D and I. Arrowheads indicate epicotyl procambium (panel E, J).
20 11-mer repeats and one 36-mer motif at the carboxyl end, with molecular mass 52 kDa and pI 6.6 (Curry and Walker-Simmons, 1993). In situ hybridization results suggested that in fresh seeds, GmPM2 transcripts are slightly detected in the vascular tissues of cotyledons and embryo axis (Figure 4B to E). In 4-day PD seeds, the signals of GmPM2 transcripts are significantly enhanced in the same regions (Figure 4G to J). As well, mesophyll and epidermis of epicotyl, cortex, and endosperm also show strong signals (Figure 4G, H, and J).
GmPM8/10 share high homology in cDNA sequences (93%). The encoded proteins contain 31 and 32 similar contiguous repeats, respectively. The molecular weights are 48 and 50 kDa, at pI 7.7 and 7.1, respectively. Both
proteins consist of 29-mer hydrophobic regions at the N terminus, which are removed after co-incubation with en-doplasmic reticulum membranes (Hsing et al., 1995). In fresh seed, GmPM8/10 transcripts (the 2 transcripts cannot be distinguished because of high identity) are detected in embryo axis, and slightly accumulated in phloem and xylem cells of vascular tissues in cotyledon (Figure 5B to F). In 4-day PD seeds, strong signals of GmPM8/10 mRNA is observed in similar tissues, and the transcripts accumulate to high levels in mesophyll and the adaxial epi­dermis of the cotyledon (Figure 5H to L). In palisade tis­sue of cotyledon, all GmPM8 transcripts aggregate around the nuclei of cells rather than being scattered around the whole cells, as for most of the other LEA messages (Figure 5I and J).
Figure 5. Localization of soybean GmPM8/10 transcripts in fresh (panel A to F) or 4-day PD (panel G to L) soybean seeds. A sense probe was used in panel A and G, and an antisense probe in the remaining panels. Bar=200 (im for panel A to C and G to I, 80 (im for panel F, 60 (im for panel D, J, and K, and 20 (im for panels E and L. Black arrowheads indicate cotyledon vascular strands (panel C, I) or epicotyl procambium (panel F). White arrowhead indicates the aggregation of GmPM8 transcripts in palisade tissue of cotyledon (panel J).
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Gm PM30 encodes a small LEA III protein, which contains five 11-mer tandem repeats. The molecular weight of GmPM30 protein is 15 kDa, at pI 9.6. In fresh seeds, the GmPM30 mRNA accumulates to a high level in the epicotyl, as well as the phloem and xylem cells in the radicle; it is also slightly detectable in cotyledon vascular tissues (Figure 6C to G). In 4-day PD seeds, GmPM30 transcripts significantly increase in vascular tissues of embryo axis and cotyledon. Strong signals are also observed in the cortex and epidermis cells of the radicle. In addition, GmPM30 transcripts are slightly enhanced in the mesophyll of the cotyledon and are highly induced in adaxial epidermis cells of the cotyledon (Figure 6G-L).
Although LEA III genes were identified from plant and animal kingdoms, few LEA III genes have been studied by in situ hybridization. Craterostigma plantag-ineum pcCAD28, encoding a D7-type protein with eleven 11-mer repeats, is a vegetative tissue-specific gene. After dehydration treatment, pcCAD28 transcripts were greatly increased in all cells of leaf or root tissues (Ditzer et al., 2001). However, the authors did not mention whether pcCAD28 transcripts were detectable in dry seeds. In the present study, the transcripts of GmPM2, GmPM8/10, or GmPM30 were mainly accumulated in leaf primordia and the radicle. As well, all studied LEA III genes were detected in vascular tissues. These results suggest that these LEA III subtypes may be responsive to dehydration in the embryo axis and conductive system.
Localization of Group IV LEA messages
Group IV LEA genes encode a group of proteins with small molecular weight. The proteins lack significant sig­nature motifs or consensus sequences as do LEA I to III proteins but contain similar amino acid residue distribu­tion along the polypeptide. They are highly hydrophilic and heat soluble (Dure, 1993; Wise, 2003). The putative open reading frame of GmPM1 (accession no. M80666) encodes a polypeptide of 173 amino acid residues, of 17.6 kDa at 10.4 pI. In situ hybridization revealed that GmPM1 transcripts accumulate in vascular tissues of embryo axis
and cotyledon in fresh seed (Figure 7B to E). In 4-day PD seeds, the mRNA expression of GmPM1 is maintained in the same region as for fresh seeds but is highly induced in vascular tissues and epidermis cells of the epicotyl (Figure 7G, H, and J) as well as xylem of cotyledon vascular strand (Figure 7I). Because the GmPM1 transcripts are relative abundant in the radicle in both fresh and PD seeds, GmPM1 genes may undergo tissue-specific regulation.
LEA IV genes have been isolated from Gymnosperm and Angiosperm. The expression patterns of these genes suggest that some of them, such as Arabidopsis PAP51 (Parcy et al., 1994) and soybean GmPM9 and GmPM16 (Lee et al., 2000; Shih et al., 2004), are seed/embryo specific, whereas C. plantagineum pcCC2 (Ditzer et al., 2001) is vegetative tissue specific. However, other LEA IV genes, such as tomato Le25 (Kahn et al., 1993) and Phaseolus vulgaris Pvlea4-25 (Colmenero-Flores et al., 1999), are expressed in seeds and embryos, as well as in vegetative tissues with stress treatments. Previous localization studies in our lab showed that the transcripts of another basic soybean LEA IV, GmPM16, accumulated in cotyledon mesophyll cells but not in the vascular system; they were also slightly detectable in the embryonic axis, especially in the metaxylem and pith tissues (Shih et al., 2004). Hence, these two LEA IV genes, GmPM1 and GmPM16, have different expression patterns.
Local i zati on of m atu rati on protei n g en e messages
GmPM5 (accession no. U59425) encodes soybean seed 7S globulin, a glycolated protein, of 41 kDa at pI 8.2. Previous work showed that GmPM5 proteins underwent post-translational modification, including removal of the signal peptide and breakage into 2 polypeptides of 27 and 16 kDa that are linked by disulfide bonds and are glycolated (Hirano et al., 1992). In situ hybridization illustrated that GmPM5 transcripts accumulate to extremely high levels in cotyledon mesophyll in both fresh and PD seeds (Figure 8B, C, E, G to I, and K). As well, no signal was found in all epidermal cells of cotyledon
Figure 6. Localization of soybean GmPM30 transcripts in fresh (panel A to G) or 4-day PD (panel H to N) soybean seeds. A sense probe was used in panel A, B, H, and I and an antisense probe in the remaining panels. Bar=200 (im for panel A to D and H to K, 80 (im for panel G and N, 60 (im for panel E and L, and 20 (im for panel F and M. Arrowheads indicate cotyledon vascular strands (panel D, K) or epicotyl procambium (panel G, N).
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Figure 7. Localization of soybean GmPM1 transcripts in fresh (panel A to E) or 4-day PD (panel F to J) soybean seeds. A sense probe was used in panel A and F and an antisense probe in the remaining panels. Bar=200 (im for panels A, B, F, and G, 80 (im for panel E and J, 60 (m for panel C and H, and 20 (m for panels D and I. Arrowheads indicate cotyledon vascular strands (panels B, G) or epicotyl procambium (panels E, J).
and embryo axis or in vascular tissues in either fresh or PD seeds (Figure 8D-E, I-J). Previous work demonstrated that GmPM5 proteins could be released into the media while the seeds were immersed in hot (40-60°C) water or were secreted into media from suspension-cultured cells (Hirano et al., 1992; Hsing, unpublished results). Extracellular dermal glycoprotein (EDGP), the carrot homolog of GmPM5, is also a secretion glycoprotein, but no post-translational cleavage was observed. In addition, the expression of EDGP genes was responsive to biotic or abiotic stresses (Satoh et al., 1992; Shang et al., 2005). Al­though these secreted glycoproteins are normally found in dermal tissues, in situ hybridization revealed no GmPM5 transcript in dermal cells. Thus, protein localization
analysis was performed by seed tissue printing. The immunoblotting results illustrated in Figure 9 indicate that although GmPM5 transcripts are not present in epidermal cells, the proteins are transported and are accumulate to high level in these cells (Lane A and B). As well, the proteins do not transport into vascular bundles (Lane E and F).
CONCLUSION
The present study characterized the spatial expression patterns of 8 soybean maturation protein genes, including 7 hydrophilic LEAs and 1 maturation protein genes, in fresh or PD seeds at 35 DAF by in situ hybridization analysis.
Figure 8. Localization of soybean GmPM5 transcripts in fresh (panel A to E) or 4-day PD (panel F to K) soybean seeds. A sense probe was used in panel A and F, and an antisense probe in the remaining panels. Bar=200 (im for panel A, B, F, and G, 80 (im for panel I, 60 (m for panel C, D, H, and J, and 20 (m for panels E and K. Black or white arrowheads indicate cotyledon vascular strands (panel B, C, G).
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Most of the hydrophilic LEA genes are expressed in vascular tissues. In fact, several LEA proteins accumulate in the conductive system of seed or vegetative tissues (e.g. Houde et al., 1995; Parra et al., 1996; Rouse et al., 1996; Danyluk et al., 1998; Arenas-Mena et al., 1999; Nylander et al., 2001; Rodrigo et al., 2004). The seed vascular sys­tem is crucial for the translocation of seed reserves from the cotyledons to the growing axis during seed germina­tion (Garnczarska et al., 2007). Hence, the presence of
LEA proteins in the vascular system of cotyledons may be required for the protection of imbibitional injury during germination. In addition, LEA proteins might also function as water attractants during the transport of water and seed reserves to sink tissue (Nylander et al., 2001; Rorat, 2006), which, as compared with other tissues, should consist of high water-uptake capacities during the early stage of im­bibition (Manz et al., 2005). This phenomenon may also be involved in the protection mechanism of germination tissues in elevating the initial moisture level (Vertucci and Leopold, 1984).
However, some hydrophilic LEA or maturation protein genes, such as GmPM16 (Shih et al., 2004) and GmPM5 (present study), were not detected in vascular tissues. Five tested LEA genes—GmPM2, GmPM6/7, GmPM8/10, GmPM12, and GmPM30―are expressed in adaxial but not abaxial cells of the cotyledon epidermis. Therefore, each LEA gene has its own tissue-specific regulation. Moreover, depending on the water status during seed maturation, seed water evaporates from the seed surface (Ishida et al., 2004; Garnczarska et al., 2007). The mesophyll or epidermis cells may sense water loss and trigger the expression of LEA genes. As well, the adaxial or abaxial epidermis of the cotyledon may display diverse responses to water potential by different sensitivity to osmotic stresses. The different spatial profiles of LEA genes reflect the large numbers of LEA genes in the plant genome. For example, on similarity
Figure 9. Localization of soybean GmPM5 proteins in 4-day PD soybean seeds by tissue printing. Protein signals of GmPM5 were detected by immunoblotting with anti-serum of GmPM5. Panel A to F indicate the blotting times 5, 15, 30, 60, 90, and 120 sec, respectively.
Figure 10. Detail localization of soybean GmPM5, 6, and 8 transcripts in 4-day PD soybean seeds. Bar=40 (im for all panels. Abbre­viations: Co, cortex; Cp, palisade tissue of cotyledon; Cs, spongy tissue of cotyledon.
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search, 50 LEA genes were detected in the Arabidopsis genome (Bies-Etheve et al., 2008). In terms of evolution, gene redundancy and different expression patterns are good strategies for survival. Therefore, a single LEA gene may not provide enough protection under different stresses, whereas many LEA families may provide such protection.
To date, although several hundreds of LEA proteins have been identified from a wide range of species, only few of them contains an ER signal peptide. GmPM5 and GmPM8/10 proteins, both containing signal peptides, target into ER membranes (Hirano et al., 1992; Hsing et al., 1995). In the present work, these 2 transcripts showed a diverse localization pattern: GmPM8/10 mRNA aggregates around nuclei whereas GmPM5 mRNA spreads out over the whole cytoplasm (Figure 10). Association of mRNAs with different ER systems was demonstrated in animal and plant systems (Melton, 1987; Svoboda, 1991; Li et al., 1993). In rice endosperm used as a model system, the transcripts of 2 different storage protein genes, prolamin and glutelin, targeted into 2 morphologically distinct ER membranes. Prolamin mRNA is highly enriched on protein-body ER, which is located near the plasma membrane, whereas glutelin mRNA is enriched at the cisternal ER that is randomly distributed throughout the cytoplasm (Krishnan et al., 1986; Yamagata and Tanaka, 1986; Kim et al., 1993; Li et al., 1993). The localization of transcripts to distinct ER membranes might be involved in a protein-sorting network (see reviews in Okita et al., 1998; Okita and Choi, 2002; Crofts et al., 2005). Thus, the different localization of GmPM5 and GmPM8/10 mRNA provides cytological evidence that ER-targeting LEA proteins may transport into distinct organelles or secretary pathways.
Acknowledgments. We thank Kaoshung Agricultural Experimental Station, Pintoung, Taiwan, for providing soybean seeds. We also express our deep thanks to Laura Smales for editing services. This research was supported by the National Science Council, ROC, and Academia Sinica, ROC, to YICH and JSH.
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大豆種子成熟蛋白基因表現的組織與細胞定位
施明德1 郭美華2 林淑琴2 徐子富1 謝金龍1
鍾美珠1 鄒稚華1 邢禹依1 謝兆樞2
1中央研究院植物暨微生物學研究所
2國立台灣大學農藝學系
        在種子成熟晚期大量表現的晚胚蛋白基因是與種子失去水份的時程有關。本研究將針對八種大豆的
GmPM 基因 ，包含七種典型與一種非典型晚胚蛋白基因，以開花後 35天的新鮮與帶莢乾燥 4天的種子
為材料，進行原位雜交的分析。結果顯示，典型的晚胚蛋白基因會在新鮮種子的子葉與胚軸的維管束中
表現。經過帶莢乾燥後，典型的晚胚蛋白基因除了在維管束中表現量會大量的增加，也會在種子的其它
組織中被誘導出來。非典型的晚胚蛋白基因，GmPM5 ，則是在新鮮與帶莢乾燥種子的非維管束組織中
大量表現。因此，晚胚蛋白基因在傳導組織的表現是受到高度調控的。此外，具有內質網訊號序列的晚
胚蛋白基因 ，GmPM5與 GmPM8/10 在細胞內的分佈也有明顯的差異。這結果顯示這些晚胚蛋白基因的
轉錄物會進入不同的內質網系統，並影響之後蛋白的轉運方式。
關鍵詞：內質網；GmPM ；原位雜交；晚胚蛋白；大豆；空間表現型式；維管束。