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. 2000 Jan;122(1):57–66. doi: 10.1104/pp.122.1.57

Multiple Mode Regulation of a Cysteine Proteinase Gene Expression in Rice1

Shin-Lon Ho 1,1,2, Wu-Fu Tong 1, Su-May Yu 1,2,*
PMCID: PMC58844  PMID: 10631249

Abstract

In many plants, cysteine proteinases play essential roles in a variety of developmental and physiological processes. In rice (Oryza sativa), REP-1 is a primary cysteine proteinase responsible for the digestion of seed storage proteins to provide nutrients to support the growth of young seedlings. In the present study, the gene encoding REP-1 was isolated, characterized, and designated as OsEP3A. An OsEP3A-specific DNA probe was used to study the effect of various factors on the expression of OsEP3A in germinating seeds and vegetative tissues of rice. The expression of OsEP3A is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen-regulated in suspension-cultured cells. The OsEP3A promoter was linked to the coding sequence of the reporter gene, gusA, which encodes β-glucuronidase (GUS), and the chimeric gene was introduced into the rice genome. The OsEP3A promoter is sufficient to confer nitrogen regulation of GUS expression in suspension-cultured cells. Histochemical studies also indicate that the OsEP3A promoter is sufficient to confer the hormonal regulation of GUS expression in germinating seeds. These studies demonstrate that in rice the REP-1 protease encoded by OsEP3A may play a role in various physiological responses and processes, and that multiple mechanisms regulate the expression of OsEP3A.

Protein degradation is an essential component in plant growth, development, and environmental responses. Selective proteolysis provides a mechanism for protein turnover and reutilization of nitrogen for maintaining cellular homeostasis and growth. Although plants can synthesize all amino acids de novo, a substantial portion of new proteins is derived from recycled amino acids (Huffaker and Peterson, 1974; Vierstra, 1993). Amino acids can be generated from the degradation of proteins or they can be derived from specialized storage proteins in seeds or vegetative tissues (Staswick, 1994). Under nutrient stress conditions, protein degradation is accelerated to maintain the supply of amino acids (Davies, 1982; Dice, 1987). Proteolysis in plants is a complex process involving many enzymes. The Cys proteinase (CysP) enzymes, with a Cys residue at the active center, are extensively studied because they appear to play a central role in a wide range of proteolytic functions in higher plants.

CysP are recognized as the major enzymes for the catabolism of the majority of reserve proteins in seeds (Shutov and Vaintraub, 1987; Ryan and Walker-Simmons, 1991). In cereal grains, nutrients are stored in the endosperm in the form of starch and storage proteins. Early in seed germination, the embryo synthesizes gibberellins (GAs), which diffuse to the aleurone cells surrounding the starchy endosperm and act as signals to activate the synthesis and secretion of several groups of hydrolytic enzymes, including α-amylases and proteases (Fincher, 1989; Ho and Hagen, 1993). Sugars, amino acids, and small peptides resulting from the hydrolysis of endosperm starch and proteins are absorbed by the embryo to support the growth of young seedlings. In barley seeds, CysPs are the primary enzymes responsible for the hydrolysis of the major storage proteins, hordeins, to small peptides (Rostogi and Oaks, 1986).

Two barley CysPs, EPA and EPB, have been purified and shown to be capable of hydrolyzing hordeins in vitro (Koehler and Ho, 1988, 1990a; Zhang and Jones, 1996). The rice (Oryza sativa) REP-1 has also been purified and characterized as a major protease for the digestion of the main seed storage protein, glutelin (Kato and Minamikawa, 1996). The expression of barley and rice CysP genes in aleurone layers is induced by gibberellic acid (GA3) and suppressed by abscisic acid (ABA) (Koehler and Ho, 1990b; Shintani et al., 1997). Recently, the promoters of two barley EPB genes were characterized and shown to confer GA induction and ABA suppression of a reporter gene expression in barley aleurone cells using transient gene expression assays (Mikkonen et al., 1996).

CysPs have also been postulated to play essential roles in many developmental and physiological processes, such as senescence and programmed cell death. mRNA levels of two CysP-related genes, SAG2 and SAG12, are significantly higher in degreening tissues than in green tissues during senescence of Arabidopsis leaves, and was proposed to be involved in the progression of senescence in somatic tissues (Hensel et al., 1993; Lohman et al., 1994). Two CysP cDNAs, tpp and TPE4A, were isolated from senescent pea ovaries. Expression of tpp significantly increases 2 to 3 DPA in unpollinated pea ovaries and was proposed to be involved in the senescence of the unpollinated ovaries (Granell et al., 1992). Expression of TPE4A is induced during the senescence of unpollinated pea ovaries and the germination of pea seeds, indicating that the same CysP may be involved in remobilization of stored nutrients in these two different physiological processes (Cercós et al., 1999). The mRNA level of a CysP gene significantly increases during in vitro tracheary element differentiation in isolated mesophyll cells of zinnia, and CysP was suggested to be involved in autolysis of cytoplasm during xylogenesis (Ye and Varner, 1996). Recently, the role of CysP in modulating programmed cell death triggered by an avirulent strain of pathogen or by oxidative stress was demonstrated by ectopic expression of a CysP inhibitor gene in cultured soybean cells (Solomon et al., 1999).

Degradation of proteins is accelerated when the tissue becomes nitrogen-limited or senescent or when the stored amino acids are needed by sink tissues, suggesting that plants have signaling pathway(s) linking the supply of free amino acids or nitrogen to the rate of intracellular proteolysis (Staswick, 1994). An interesting question is thus raised regarding how the degradation of proteins is regulated. In the present study, the control mechanism of protease gene expression in plants was explored. As a first step, we investigated various factors that regulate the expression of a CysP in rice. We found that expression of a CysP gene, OsEP3A, in rice is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen regulated in suspension-cultured cells. We also found that the promoter of OsEP3A confers GA3 induction of reporter gene expression in transformed germinating rice seeds. The same promoter also confers nitrogen-deprivation-induced expression of reporter gene expression in transformed rice suspension-cultured cells. To our knowledge, this is the first report of multiple modes of regulation of a single proteinase gene expression in plants.

MATERIALS AND METHODS

Plant Material

The rice variety used in this study was Oryza sativa L. cv Tainung 67. Immature seeds were dehulled, sterilized with 2.4% NaOCl for 1 h, washed extensively with sterile water, and placed on N6D agar medium (Toki, 1997) for callus induction. After 1 month, callus derived from scutella was subcultured in fresh N6D medium for transformation or to a liquid Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 3% Suc and 10 μm 2,4-dichlorophenoxyacetic acid to establish a suspension cell culture as previously described (Yu et al., 1991).

Screening of Rice Genomic Library and DNA Sequence Analysis

Approximately 2 × 106 recombinant λ phage clones derived from a rice (cv Japonica) genomic DNA library (CLONTECH Laboratories, Palo Alto, CA) were screened. Plaques were lifted on nylon transfer membranes (MSI, Westborough, MA) and probed with 32P random-primer-labeled cDNA of EP-B, pHVEP4 (Koehler and Ho, 1990b). One positive clone containing a 2.6-kb insert was selected and subcloned into the EcoRI site of pBluescript KS+ (Stratagene, La Jolla, CA) and designated as pOsEP3A. The complete nucleotide sequence of the rice Cys endoprotease gene OsEP3A was determined from both orientations with an automated laser fluorescent DNA sequencer (Pharmacia, Piscataway, NJ) and by the manual dideoxy chain-termination method (Sequenase version 2, United States Biochemical, Cleveland). DNA sequence analysis was performed using the University of Wisconsin Genetics Computer Group software package, version 9.1.

Plasmid Constructions

Plasmid pRY18 carries a 3.8-kb DNA fragment that contains a rice genomic rDNA cluster, including the 3′ half portion of the 17S rRNA gene, the complete 5.8 S rRNA gene, and the 5′ half portion of the 25S rRNA gene in pUC13 (Sano and Sano, 1990). A 1.1-kb DNA fragment containing the 5′ flanking region and the first 28 amino acids of OsEP3A was PCR-amplified using the T7 primer (Stratagene) and the primer 5′-GATATCTGCAGGGTATCGCGGCGCACAG-3′ (sequence complementary to positions +195 to +211 of OsEP3A underlined). This DNA fragment was cleaved with PstI at both ends and cloned into the PstI site of pBX-2 in such a way as to allow an in-frame fusion with the gusA (Jefferson, 1987) coding region. pBX-2, a pBluescript KSII+ (Stratagene)-derived plasmid, containing the gusA gene and a nopaline synthase gene (Nos) polyadenylation site between the BamHI and XbaI sites was kindly provided by Dr. Ray Wu (Cornell University, Ithaca). A DNA fragment containing the cauliflower mosaic virus 35S RNA gene (35S) promoter-hygromycin phosphotransferase coding region (Hph)-tumor morphology large gene terminator (Tml 3′) was excised from pTRA151 (Zheng et al., 1991) with PstI and EcoRI and inserted into the same sites in pPZP200 (Hajdukiewicz et al., 1994) to generate pPZP200-H. The virG gene of Agrobacterium tumefaciens pTiBo542 (Chen et al., 1991) was PCR-amplified with blunt ends and inserted into the ScaI site of pPZP200-H to generate pSMY1H. The OsEP3A promoter-gusA-Nos3′ chimeric gene was excised from pBX-2 with HindIII and inserted into the HindIII site of pSMY1H to generate pEPGS1.

Primer Extension Analysis

5′-Primer extension analysis was performed according to the method of Sambrook et al. (1989). Total RNA was isolated from germinating embryos 3 d after germination. Poly(A+) RNA was purified from the total RNA and hybridized with 32P-labeled primer P2 (5′-ATCGATCGATCGCCACT-3′) (Fig. 1). The polymerization reaction was conducted with reverse transcriptase (RT) (SuperScript, GIBCO-BRL, Cleveland). Dideoxynucleotide sequencing of OsEP3A was also performed using the P2 primer and served as the sequence reference. The extension product and the sequence references were electrophoresed and visualized by autoradiography.

Figure 1.

Figure 1

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Nucleotide and deduced amino acid sequences of OsEP3A. The 5′- and 3′-flanking regions are shown in lowercase letters. The coding region is shown in uppercase letters. Nucleotide +1 is the mapped transcription start site. The predicted signal peptide sequence is underlined. The putative TATA box and polyadenylation signals are double-underlined. The putative GARE is labeled with bold underline. The positions of PCR primers used for examination of intron (P1 and P3) and for synthesis of OsEP3A-specific probe (P1 and P2) are labeled with arrows. The nucleotide sequence of OsEP3A is in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number AF099203.

PCR

DNA PCR of genomic clone pOsEP3A was performed using oligonucleotides P1 (5′-ATCGCCCTCACCCTCCA-3′, positions +1 to +17) and P3 (5′-TGTACGGCGGAGATGGC-3′, positions +1240 to to +1256) (Fig. 1) as primers. The PCR product, P1–3, containing the coding region of OsEP3A was later used as a probe in genomic DNA Southern-blot analysis. Seed mRNA RT-PCR was performed using poly(A+) RNA isolated from germinating embryos as a template and oligonucleotides P1 and P3 as primers according to methods previously described (Chan and Yu, 1998). PCR using pOsEP3A DNA as a template and oligonucleotides P1 and P2 as primers generated a 120-bp DNA fragment P1–2. P1–2,, which contains the 5′-untranslated region (5′UTR) of OsEP3A, was later used as a gene-specific probe in genomic DNA Southern-blot and RNA northern-blot analyses.

Genomic DNA Southern-Blot Analysis

Genomic DNA was isolated from wild-type or transformed calli according to the method of Sheu et al. (1996). Ten micrograms of genomic DNA was digested with restriction enzymes, fractionated in a 0.8% agarose gel, and transferred to a nylon membrane (MSI). Hybridization was performed at 42°C using 32P random-primer-labeled OsEP3A cDNA (P1–3) or gene-specific DNA (P1–2) as a probe.

Northern-Blot Analysis

Total RNA was isolated from various tissues of germinating seeds according to the method of Yu et al. (1996) and isolated from suspension-cultured cells using TRIZOL reagent (GIBCO-BRL). RNA gel-blot analysis was performed as described by Thomas (1983). Ten micrograms of total RNA was electrophoresed in 1% agarose gel containing 10 mm sodium phosphate buffer (pH 6.5), transferred to a nylon filter, and hybridized with 32P random-primer-labeled P1–2 or rDNA probe. The blot was visualized using autoradiography and quantified using phosphor-imaging software (PhosphoImager, Molecular Dynamics, Sunnyvale, CA).

Transformation

Plasmid pEPGS1 was introduced into A. tumefaciens strain EHA101 (Hood et al., 1986) with an electroporator (BTX, San Diego) according to the manufacturer's instructions. Calli induced from immature rice seeds were co-cultured with A. tumefaciens according to the methods described by Hiei et al. (1994) and Toki (1997).

Western-Blot Analysis

Total proteins were extracted from suspension-cultured cells with an extraction buffer (50 mm Tris-HCl, pH 8.8, 1 mm EDTA, 10% [v/v] glycerol, 1% [v/v] Triton X-100, 10 mm β-mercaptoethanol, and 0.1% [w/v] sarkosyl). The culture medium was collected and centrifuged at 18,000g at 4°C for 15 min to remove cell debris. Western-blot analysis was performed as described by Yu et al. (1991). The GUS polyclonal antibodies (Molecular Probes, Eugene, OR) were diluted 10,000-fold prior to use.

Histochemical Localization of GUS Activity

The dehulled seeds with or without embryos were sterilized with 2.4% NaOCl and placed on two pieces of sterile Whatman No. 1 filter paper in a Petri dish. A solution containing 1 μm GA3 and 10 mm CaCl2 or sterile water was applied to the filter paper to imbibe the seeds. The seeds were incubated at 28°C in darkness for various time periods. After incubation, seeds were hand-cut longitudinally in half and stained with 1 mm 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-gluc) at 37°C in darkness for 12 h. The stained seeds were reserved in 70% ethanol and rinsed with water before photography.

RESULTS

Cloning and Characterization of a Rice CysP Gene, OsEP3A

To elucidate the hormonal, developmental, and metabolic factor regulating rice CysP gene expression, we cloned and characterized a rice CysP gene. A rice gene that hybridized strongly with the barley CysP cDNA clone pHVEP4 was isolated and designated as OsEP3A. The OsEP3A clone containing 2588 bp was sequence analyzed. The complete coding and the 5′- and 3′-flanking sequences of OsEP3A are shown in Figure 1. Comparison with sequence data in GenBank showed that the coding sequence of OsEP3A matches perfectly with rice pRP60 cDNA, which encodes REP-1 (Kato and Minamikawa, 1996). The intronless feature of OsEP3A is similar to the barley EPB genes (Mikkonen et al., 1996). To verify that OsEP3A is intronless, oligonucleotides P1 and P3 (Fig. 1) were used as primers for genomic DNA PCR and seed mRNA RT-PCR analyses. A 1.26-kb DNA fragment was amplified from both genomic DNA and mRNA, which confirmed that OsEP3A was intronless (data not shown). The transcription start site was mapped to be located at the adenosine nucleotide 122 bp upstream from the initiation codon (data not shown) and designated as +1. A typical TATA box is located at –34 of the transcription start site (Fig. 1).

DNA-blot analysis was performed to determine the copy number of CysP gene in the rice genome. By using DNA fragment P1–3 as a probe, under very low hybridization stringency, one strong hybridization band and some minor bands were observed irrespective of the restriction enzymes used (Fig. 2, lanes 1–3). This finding indicated that the rice CysPs are encoded by a multigene family. By using DNA fragment P1–2 as a probe, only a single band was hybridized (Fig. 2, lanes 4–6). Nucleotide sequence analysis revealed that no significant homology was present among the 5′UTR of OsEP3A and four other rice CysP cDNAs (Watanabe et al., 1991; Shintani et al., 1997). It is very likely that OsEP3A exists as one-copy gene in the rice genome. Therefore, the 5′UTR of OsEP3A was served as a gene-specific probe.

Figure 2.

Figure 2

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Gel-blot analyses of rice genomic DNA showing that CysP are encoded by a gene family. Rice genomic DNA was digested with BamHI (B), EcoRI (E), or PstI (P) and subjected to gel-blot analysis using the full-length OsEP3A cDNA (lanes 1–3) or P1–2 (lanes 4–6) as a probe. The molecular mass markers are shown in kb on the left of the autoradiogram.

Temporal and Spatial Regulation of OsEP3A Expression in Rice

To examine the expression pattern of OsEP3A in germinating seeds, total RNA was purified from embryos and endosperms and subjected to gel-blot analysis using P1–2 as a probe. As shown in Figure 3A, OsEP3A mRNA was barely detectable in the embryos of dry seeds (lane 1), became detectable 1 h after imbibition (lane 2), and then gradually increased with incubation time (lanes 3–7). Gel-blot analysis of RNA accumulated in the embryos and endosperms showed that levels of OsEP3A mRNA fluctuated during a 10-d seed germination period. The levels of OsEP3A mRNA in embryos (Fig. 3B) and endosperms (Fig. 3C) reached their first peaks at d 4, declined from d 5 to d 7, then increased again and reached their second peaks at d 8 to 9 after the onset of seed germination.

Figure 3.

Figure 3

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Developmental expression of OsEP3A in germinating rice seeds. Rice seeds were germinated for various time periods. Total RNA was purified from the embryos and endosperms and subjected to RNA gel-blot analysis using P1–2 or rDNA gene as a probe. Ten micrograms of total RNA was loaded in each lane. A, Levels of mRNA in embryos germinated for 48 h. B, Levels of mRNA in embryos germinated for 10 d. C, Levels of mRNA in endosperms of seeds germinated for 10 d. The experiment was performed twice producing similar result.

Total RNA was also purified from various vegetative tissues of rice at different growth stages and subjected to gel-blot analysis using P1–2 as a probe. As shown in Figure 4, the OsEP3A mRNA was barely detectable in the shoot (lane 1) and root (lane 2) of a 20-d-old seedling and in the stem (lane 4) and sheath (lane 7) of a 3-month-old mature plant. OsEP3A mRNA level was low in the root (Fig. 4, lane 3) but high in the green leaves (Fig. 4, lane 5) and senescing leaves (Fig. 4, lane 6) of the mature plant. Quantitation of mRNA indicates that the level of OsEP3A mRNA in the mature green leaves and senescing leaves was 3- and 9-fold, respectively, of that in mature root. These studies demonstrate that the expression of OsEP3A in vegetative tissues is developmentally and spatially regulated.

Figure 4.

Figure 4

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Developmental and spatial expression of OsEP3A in rice plants. Various tissues of young (20-d-old) and mature (3-month-old) plants were collected and total RNA was purified and subjected to RNA gel-blot analysis using P1–2 or rDNA gene as a probe. S, Shoot; R, root; St, stem; L, leaf; SL, senescencing leaf; Sh, sheath.

Metabolic Regulation of OsEP3A Expression in Suspension-Cultured Rice Cells

Plant cells store nitrogen in the form of proteins, and degradation of proteins allows recycling of amino acids and nitrogen. To investigate whether the expression of OsEP3A is regulated by nitrogen, rice cells were cultured in MS medium with or without nitrogen sources (NH4NO3 plus KNO3) for various time periods. Total RNA was purified and subjected to gel-blot analysis using P1–2 as a probe. OsEP3A mRNA was not detectable in cells provided with nitrogen but accumulated in cells starved of nitrogen (Fig. 5A). The accumulation of OsEP3A mRNA was detected 1 d after nitrogen starvation and increased gradually up to 12 d, at which point the experiment was terminated. The accumulation of OsEP3A mRNA was suppressed by the addition of nitrogen sources into the culture medium and became undetectable within 48 h (Fig. 5B). These results indicate that expression of OsEP3A is suppressed by nitrogen and activated by nitrogen starvation.

Figure 5.

Figure 5

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Nitrogen starvation activates expression of OsEP3A in rice suspension cells. A, Rice suspension cells were cultured in complete MS medium (containing 20 mm each of NH4NO3 and KNO3) for 4 d and transferred to MS medium with (+) or without (−) nitrogen sources (N) and cultured for 12 d. B, Cells cultured in nitrogen-free medium for 12 d were transferred into complete MS medium for 48 h. Total RNA was purified from cells and subjected to gel-blot analysis using P1–2 or rDNA as a probe.

To determine whether the expression of OsEP3A was specifically induced by nitrogen starvation and affected by different forms of nitrogen source, rice suspension cells were cultured in medium with or without Suc or different sources of nitrogen. Within an 8-d culture period, the accumulation of OsEP3A mRNA was not or barely detectable in cells cultured in complete MS medium (containing NH4NO3 plus KNO3) with Suc (Fig. 6A, lane 1) or MS medium without Suc (Fig. 6A, lane 2), but increased significantly in MS medium without any nitrogen source (Fig. 6A, lane 3). The accumulation of OsEP3A mRNA was also not detectable in MS medium with either NH4NO3 or KNO3 (Fig. 6A, lanes 4 and 5) as the nitrogen source. These results demonstrate that expression of OsEP3A is specifically induced by nitrogen starvation and not by Suc starvation. The accumulation of OsEP3A mRNA in the nitrogen-starved cells (Fig. 6B, lane 1) could be significantly repressed by the addition of Asn, Gln, Glu, NH4Cl, or NH4NO3 into the MS medium as a nitrogen source (Fig. 6B, lanes 2–6). The addition of KNO3 into the MS medium as a nitrogen source partially suppressed the accumulation of OsEP3A mRNA (Fig. 6B, lane 7), further demonstrating that a metabolizable nitrogen source may suppress the expression of OsEP3A.

Figure 6.

Figure 6

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Nitrogen specifically suppresses the expression of OsEP3A. A, Rice suspension cells were cultured in complete MS medium (RNA in lane 1), MS medium without Suc (RNA in lane 2) or a nitrogen source (RNA in lane 3), or MS medium containing 20 mm of NH4NO3 (RNA in lane 4) or 20 mm KNO3 (RNA in lane 5) as the sole nitrogen source for 8 d. B, Cells starved of nitrogen for 8 d were transferred to fresh MS medium without a nitrogen source (RNA in lane 1) or fresh MS medium containing 10 mm each of Asn, Gln, or Glu, or 20 mm each of NH4Cl, NH4NO3, or KNO3 (lanes 2–7) for 2 d. Total RNA was purified from cells and subjected to gel-blot analysis using P1–2 or rDNA gene as a probe.

Analysis of OsEP3A Promoter Activity in Transformed Rice Suspension Cells

To investigate the role of the promoter in the regulation of OsEP3A expression in rice, a 1.1-kb DNA fragment containing the 5′ regulatory and putative signal peptide sequences of OsEP3A was fused in-frame at the 5′ end of gusA gene. The chimeric gene was inserted into a binary vector to generate pEPGS1 as shown in Figure 7A. pEPGS1 was introduced into A. tumefaciens for rice transformation. Thirty transgenic lines were regenerated and four lines containing a single copy of the gusA gene (data not shown) were selected for further study. Callus derived from scutella of transgenic seeds was cultured as suspension cells. The transformed suspension cells were then cultured in medium with or without nitrogen. Total RNA was purified and subjected to gel-blot analysis using the gusA cDNA plus P1–2 as probes. Accumulation of both gusA and OsEP3A mRNAs was detectable in cells starved of nitrogen but not in cells provided with nitrogen (Fig. 7B, lanes 3–10). No gusA mRNA was detected in the non-transformed cells (Fig. 7B, lanes 1 and 2).

Figure 7.

Figure 7

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The OsEP3A promoter confers nitrogen-starvation-activated expression in transformed rice suspension cells. Calli were generated from T1 transgenic rice seeds containing the OsEP3A-gusA chimeric gene and cultured as suspension cells. The transformed suspension cells were cultured in MS medium with (+) or without (−) a nitrogen source (N) for 10 d. A, Schematic diagram of the plasmid pEPGS1 used for rice transformation. The promoter region and signal peptide (sp) sequence (the latter underlined) of OsEP3A were in-frame joined to the coding region of gusA with the polyadenylation signals of the nopaline synthase gene (nos). RB and LB, Right and left border of T-DNA, respectively; 35S, cauliflower mosaic virus 35S RNA promoter; hph, hygromycin phosphotransferase gene; tml, turmor morphology large gene terminator; ATG, start of the gusA coding region. B, Total RNA was purified from treated cells and subjected to gel-blot analysis using gusA cDNA plus P1–2 or rDNA as probes. C, Total proteins were extracted from transformed cells or collected from the culture medium of transgenic line 14 and subjected to western-blot analysis using the GUS antibodies. One-hundred-twenty microliters of culture medium was applied in each of lanes 1 to 4. Twenty micrograms of total cellular proteins were applied in each of lanes 5 to 8. NT, Non-transformed control cell line. Lane 9 contains 200 ng of purified Escherichia coli GUS.

The levels of OsEP3A mRNA in the four transgenic lines were fairly similar, while the levels of gusA mRNA varied from line to line, indicating a position effect on transgene expression. Suspension-cultured cells of transgenic line 14 were selected for further study of the function of the putative signal peptide sequence in protein secretion. Cells were grown in medium with or without nitrogen for 10 d and proteins were extracted from cells or collected from the culture medium and subjected to western-blot analysis using GUS antibodies. As shown in Figure 7C, GUS was detected in cells and medium only when cells were starved of nitrogen. This result indicates that the putative signal peptide of OsEP3A is capable of directing translocation of GUS through the secretory pathway of transformed cells into the culture medium.

Analysis of OsEP3A Promoter Activity in Transgenic Rice Seeds

To investigate the role of promoter in the temporal, spatial, and hormonal regulation of OsEP3A expression during rice seed germination, histochemical GUS assays of transgenic seeds carrying OsEP3A-gusA chimeric gene were performed. As shown in Figure 8A, in longitudinally cut germinating seeds, GUS activity was detected in the scutellar epithelium within 1 d after germination (panel I), spread into the adjacent aleurone layer by d 3 (panels II and III), and finally covered the entire aleurone layer by d 5 (panels IV and V). As shown in Figure 8B, in cross-cut germinating seeds, GUS activity was not detected in the non-transformed aleurone layer by d 3 (panel I), but was first detected in the ventral side of the aleurone layer within 1 d after germination (panel II), spread into the dorsal side of aleurone layer by d 3 (panel III), and finally almost the entire aleurone layer by d 5 (panel IV).

Figure 8.

Figure 8

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Histochemical analysis of temporal, spatial, and hormonal regulation of OsEP3A-gusA expression in transgenic rice seeds. A, Transgenic seeds were germinated in water at 28°C for various time periods, longitudinally cut in half, and stained for GUS activity. Shoots were removed before staining. I, II, and IV are viewed from the cutting surface and III and V are viewed from the grain surface. II and III are the same half-seed and IV and V are the same half-seed. B, Germinating transgenic seeds were cross-cut in half and stained for GUS activity. I, Non-transformed seed; II to IV, transformed seeds. The dorsal side of the half-seeds is positioned up. C, Transgenic rice seeds were de-embryonated and incubated in a solution containing 1 μm GA3 and 10 mm CaCl2 at 28°C for various time periods. The non-transformed seed (I) was similarly treated for 3 d. The treated seeds were longitudinally cut in half, then stained for GUS activity. I, II, and III are viewed from the cutting surface, and IV and V are viewed from the grain surface. III and IV are the same half-seed and V is an uncut seed.

To examine the effect of GA3 on the expression of OsEP3A-gusA gene in seeds, de-embryonated transgenic rice seeds were treated with GA3 for various time periods and histochemical GUS assays were performed. As shown in Figure 8C, in the GA3-treated and longitudinally cut seeds, GUS activity was not detected in the non-transformed de-embryonated seed by d 3 (panel I), but was first detected in the ventral side of the aleurone layer within 1 d (panel II), in both the ventral and dorsal sides of the aleurone layer by d 3 (panels III and IV), and finally in the entire aleurone layer by d 5 (panel V).

DISCUSSION

The rice REP-1 cDNA has been isolated and characterized and the deduced amino acid sequence shares 75% similarity with that of the barley EPB (Kato and Minamikawa, 1996). Since REP-1 plays an essential role in the degradation of seed storage proteins during germination and early seedling growth, it is important to study how the expression of REP-1 is regulated. As a first step, we isolated the rice gene, OsEP3A, encoding REP-1. Genomic DNA-blot analysis showed that the rice CysP gene family consists of only one member of OsEP3A and that at least five other CysP gene family members have sequences similar to OsEP3A (Fig. 2). Three other rice CysP cDNAs encoding oryzains α, β, and γ (Watanabe et al., 1991) and another rice CysP cDNA pRP80 (Shintani et al., 1997) have also been isolated from the cDNA library of germinating rice seeds. The OsEP3A (pRP60) and pRP80 DNAs do not cross-hybridize to each other (Shintani et al., 1997), and the nucleotide sequence of OsEP3A coding region shares 58%, 56%, and 50% homology with oryzains α, β, and γ cDNAs, respectively. Comparison of nucleotide sequences in promoter regions reveals that sequence +18 to −120 of OsEP3A shares 86% homology with sequence +18 to −180 (containing 60-bp extra non-conserved sequences within this region) of the barley EPB-1 and EPB-2 (Mikkonen et al., 1996), but sequences upstream of this regions show very low homology between OsEP3A and EPB.

The OsEP3A-specific DNA probe was useful for studying the expression pattern of OsEP3A in germinating seeds and other tissues of rice. In germinating rice seeds, the amount of OsEP3A mRNA fluctuate in both the embryos (Fig. 3B) and endosperms (Fig. 3C). The temporal and fluctuant expression pattern of OsEP3A is similar to that of α-amylase genes (Yu et al., 1996), suggesting that expression of OsEP3A and α-amylase genes may be subject to similar regulation by both GA3 and osmotica in the rice embryo and endosperm (Yu et al., 1996). OsEP3A is also expressed in other rice tissues, particularly in the roots and leaves of mature plant and in senescing leaves (Fig. 5). Nitrogen remobilization is one of the major functions of senescence and is achieved through the action of proteolytic enzymes (Staswick, 1994). Expression of CysP genes increases in senescent leaves of various plant species and is considered to be responsible for protein degradation (Hensel et al., 1993; Lohman et al., 1994; Xu and Chye, 1999). In the present study, accumulation of OsEP3A mRNA in mature and senescing leaves is consistent with the role of OsEP3A in protein turnover and nitrogen remobilization. Interestingly, OsEP3A seems to degrade both seed and vegetative storage proteins.

Our data showed that expression of OsEP3A in suspension-cultured rice cells was induced under conditions of nitrogen deprivation. Questions were thus raised regarding the in vivo role of OsEP3A in the nitrogen-starved cells. Cells store nitrogen in the form of proteins and nitrogen deprivation may trigger degradation of cellular proteins. The amino acids and nitrogen released then contribute to the pool of soluble nitrogen for re-utilization and synthesis of new proteins under nitrogen starvation. Consequently, the synthesis of proteinase such as OsEP3A could be crucial for cell survival. On the other hand, under normal growth conditions with an adequate supply of nitrogen source, expression of OsEP3A is subject to metabolic repression. For various metabolizable nitrogen sources tested, all except KNO3 significantly suppressed the accumulation of OsEP3A mRNA. Provision of KNO3 to the nitrogen-starved cells only partially suppressed the accumulation of OsEP3A mRNA (Fig. 6B, lane 7), which could be due to a slower rate in the uptake of NO3− by the nitrogen-starved cells. It has been shown that roots of intact rice plants have a similar affinity for NH4+ and NO3−, however, the uptake rate is somewhat higher for NH4+ than for NO3− (Youngdahl et al., 1982). In suspension-cultured rice cells, the expression pattern of OsEP3A induced by nitrogen starvation is similar to that of α-amylase genes induced by sugar starvation (Sheu et al., 1994). One major difference is that cultured cells die within 5 d after Suc starvation (Chen et al., 1994), whereas cells remain alive and continue to express OsEP3A 10 to 12 d after nitrogen starvation (Fig. 5A). Such phenomena reflect a much higher demand for carbohydrates than nitrogen in the maintenance of cell life.

Our study demonstrates that expression of the OsEP3A-gusA chimeric gene in transformed rice suspension cells is induced by nitrogen starvation. This finding indicates that the 1.2-kb OsEP3A promoter region contains the necessary cis-regulatory sequences for conferring metabolic regulation of downstream gene expression. This promoter sequence also confers GA3 regulation of OsEP3A expression in germinating seeds. These results suggest that the nitrogen and GA3 regulations both act at the level of transcription. The putative 28-amino acid signal sequence predicted with a statistical method (Von Heijne, 1986) is capable of directing extracellular targeting of GUS into the culture medium of suspension cells, as well as secretion of GUS from aleurone cells into the starchy endosperm.

Histochemical GUS assays of germinating transgenic seeds revealed patterns of temporal and spatial regulation of the OsEP3A-gusA gene, which is consistent with studies using RNA gel-blot analysis (Fig. 3). The temporal and spatial expression of OsEP3A-gusA is similar to that of the Ramy1A (an α-amylase gene)-gusA gene in germinating transgenic rice seeds (Itoh et al., 1995). Furthermore, these two chimeric genes show a similar pattern of response to GA3 in aleurone layers of embryoless seeds. The absence of GUS activity in the region of the aleurone layer that surrounds the wound site where embryos and scutella were removed (Fig. 8C) was probably due to death of cells in this region during sterilization of the embryoless transgenic seeds by NaOCl prior to treatment with GA3. It is interesting to note that accumulation of GUS is first detected in the scutellar epithelium of embryo, then the proximal region of the ventral side of aleurone layer, and finally the entire aleurone layer. Such pattern of GUS accumulation indicates progression pattern of storage protein breakdown in the endosperm of germinating rice seeds.

It appears that OsEP3A and α-amylase genes are coordinately regulated by GA3 and the promoters of the two sets of genes may share common features essential for regulation of gene expression. A GA-response element (GARE) is essential for GA-dependent expression of several α-amylase genes in barley aleurone cells (Skriver et al., 1991; Gubler and Jacobsen, 1992; Lanahan et al., 1992). An MYB transcription factor, HvGAMYB, binds to the GARE of a barley high-pI α-amylase gene promoter and is able to transactivate the high pI α-amylase gene promoter in transient expression assays (Gubler et al., 1995). Two sequences resemble the consensus HvGAMYB binding site (Gubler et al., 1999) are located at positions between −163 to −156 and −143 to −136 of the OsEP3A promoter (Fig. 1) and one or both may function as a GARE. Identification of the nitrogen- and senescence-responsive cis-regulatory elements in the OsEP3A promoter is yet to be determined, and will be required to elucidate the mechanisms underlying the regulation of OsEP3A expression under different physiological conditions.

In conclusion, the transgenic approach will be useful in studying functions and interactions of cis-regulatory elements in the OsEP3A promoter that are essential for GA and temporal and spatial regulation in germinating cereal grains. In addition, nitrogen-starved suspension cells express OsEP3A in a manner similar to senescencing leaves in terms of nitrogen demand and re-utilization. Because the expression of CysP and the degradation of proteins in cultured cells can be easily manipulated by altering the nitrogen level in the medium, the rice cell culture system represents an ideal tool for studying the biochemical and molecular biological aspects of CysP gene regulation, nitrogen metabolism, and senescence in plants.

ACKNOWLEDGMENT

We thank Lin-Tze Yu for technical assistance.

Footnotes

1

This work was supported by a grant from the National Science Council of the Republic of China (no. NSC81–0211–B–003–504 to W.-F.T.) and a grant from Academia Sinica of the Republic of China (to S.-M.Y.).

LITERATURE CITED

  1. Cercós M, Santamaría S, Carbonell J. Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea. Plant Physiol. 1999;119:1341–1348. doi: 10.1104/pp.119.4.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chan M-T, Yu S-M. The 3′ untranslated region of a rice α-amylase gene mediate sugar-dependent abundance of mRNA. Plant J. 1998;15:685–696. doi: 10.1046/j.1365-313x.1998.00244.x. [DOI] [PubMed] [Google Scholar]
  3. Chen CY, Wang L, Winans SC. Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542. Mol Gen Genet. 1991;230:302–309. doi: 10.1007/BF00290681. [DOI] [PubMed] [Google Scholar]
  4. Chen M-H, Liu L-F, Chen Y-R, Wu H-K, Yu S-M. Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells are coordinately regulated by sugar nutrient. Plant J. 1994;6:625–636. doi: 10.1046/j.1365-313x.1994.6050625.x. [DOI] [PubMed] [Google Scholar]
  5. Davies DD. Physiological aspects of protein turnover. In: Coulter D, Partier B, editors. Encyclopedia of Plant Physiology. 14A. Berlin: Springer-Verlag; 1982. pp. 189–228. [Google Scholar]
  6. Dice JF. Molecular determinants of protein half-lives in eukaryotic cells. FASEB J. 1987;1:349–357. doi: 10.1096/fasebj.1.5.2824267. [DOI] [PubMed] [Google Scholar]
  7. Fincher GB. Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu Rev Plant Physiol Plant Mol Biol. 1989;40:305–346. [Google Scholar]
  8. Granell A, Harris N, Pisabarro AG, Carbonell J. Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin. Plant J. 1992;2:907–915. doi: 10.1046/j.1365-313x.1992.t01-5-00999.x. [DOI] [PubMed] [Google Scholar]
  9. Gubler F, Jacobsen JV. Gibberellin-responsive elements in the promoter of a barley high-PI α-amylase gene. Plant Cell. 1992;4:1435–1441. doi: 10.1105/tpc.4.11.1435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gubler F, Kalla R, Roberts JK, Jacobsen JV. Gibberellin-regulated expression of a myb gene in barley aleurone cells: evidence for Myb transactivation of a high-pI α-amylase gene promoter. Plant Cell. 1995;7:1879–1891. doi: 10.1105/tpc.7.11.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gubler F, Raventos D, Keys M, Watts R, Mundy J, Jacobsen JV. Target genes and regulatory domains of the GAMYB transcriptional activator in cereal aleurone. Plant J. 1999;17:1–9. doi: 10.1046/j.1365-313x.1999.00346.x. [DOI] [PubMed] [Google Scholar]
  12. Hajdukiewicz P, Svab Z, Maliga P. The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 1994;25:989–994. doi: 10.1007/BF00014672. [DOI] [PubMed] [Google Scholar]
  13. Hensel HL, Grbić V, Baumgarten DA, Bleecker AB. Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell. 1993;5:553–564. doi: 10.1105/tpc.5.5.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994;6:271–282. doi: 10.1046/j.1365-313x.1994.6020271.x. [DOI] [PubMed] [Google Scholar]
  15. Ho T-HD, Hagen G. Hormonal regulation of gene expression. J Plant Growth Regul. 1993;12:197–205. [Google Scholar]
  16. Hood EE, Helmer GL, Fraley RT, Chilton M-D. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol. 1986;168:1291–1301. doi: 10.1128/jb.168.3.1291-1301.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Huffaker RC, Peterson LS. Protein turnover in plants and possible means of its regulation. Annu Rev Plant Physiol. 1974;25:363–392. [Google Scholar]
  18. Itoh K, Yamaguchi J, Huang N, Rodriguez RL, Akazawa T, Shimamoto K. Developmental and hormonal regulation of rice α-amylase (Ramy1A)-gusA fusion genes in transgenic rice seeds. Plant Physiol. 1995;107:25–31. doi: 10.1104/pp.107.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jefferson RA. Assaying chimeric genes in plants from gene fusion system. Plant Mol Biol Rep. 1987;5:387–405. [Google Scholar]
  20. Kato H, Minamikawa T. Identification and characterization of a rice cysteine endopeptidase that digests glutelin. Eur J Biochem. 1996;239:310–316. doi: 10.1111/j.1432-1033.1996.0310u.x. [DOI] [PubMed] [Google Scholar]
  21. Koehler SM, Ho T-HD. Purification and characterization of gibberellic acid-induced cysteine endoproteases in barley aleurone layers. Plant Physiol. 1988;87:95–103. doi: 10.1104/pp.87.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Koehler SM, Ho T-HD. A major gibberellic acid-induced barley cysteine proteinase which digests hordein. Plant Physiol. 1990a;94:251–258. doi: 10.1104/pp.94.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Koehler SM, Ho T-HD. Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers. Plant Cell. 1990b;2:769–783. doi: 10.1105/tpc.2.8.769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lanahan MB, Ho T-HD, Rogers SW, Roger JC. A gibberellin response complex in cereal α-amylase gene promoters. Plant Cell. 1992;4:203–211. doi: 10.1105/tpc.4.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lohman KN, Gan S, John MC, Amasino RM. Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant. 1994;92:322–328. [Google Scholar]
  26. Mikkonen A, Porali I, Cercos M, Ho T-HD. A major cysteine proteinase, EPB, in germinating barley seeds: structure of two intronless genes and regulation of expression. Plant Mol Biol. 1996;31:239–254. doi: 10.1007/BF00021787. [DOI] [PubMed] [Google Scholar]
  27. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 1962;15:473–497. [Google Scholar]
  28. Rostogi V, Oaks A. Hydrolysis of storage proteins in barley endosperms. Plant Physiol. 1986;81:901–906. doi: 10.1104/pp.81.3.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ryan C, Walker-Simmons M. Plant proteinases. In: Stumpf PK, Conn EE, editors. The Biochemistry of Plants: A Comprehensive Treatise. 6: Proteins and Nucleic Acids. New York: Academic Press; 1991. pp. 321–350. [Google Scholar]
  30. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Ed 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  31. Sano Y, Sano R. Variation of the intergenic spacer region of ribosomal DNA in cultivated and wild rice species. Genome. 1990;33:209–218. [Google Scholar]
  32. Sheu J-J, Jan S-P, Lee H-T, Yu S-M. Control of transcription and mRNA turnover as mechanisms of metabolic regulation in rice α-amylase gene expression. Plant J. 1994;5:655–664. [Google Scholar]
  33. Sheu J-J, Yu T-S, Tong WF, Yu S-M. Carbohydrate starvation stimulates differential expression of rice α-amylase genes that is modulated through complicated transcriptional and posttranscriptional processes. J Biol Chem. 1996;271:26998–27004. doi: 10.1074/jbc.271.43.26998. [DOI] [PubMed] [Google Scholar]
  34. Shintani A, Kato H, Minamikawa T. Hormonal regulation of expression of two cysteine endopeptidase genes in rice seedlings. Plant Cell Physiol. 1997;38:1242–1248. doi: 10.1093/oxfordjournals.pcp.a029111. [DOI] [PubMed] [Google Scholar]
  35. Shutov AD, Vaintraub JA. Degradation of storage proteins in germinating seeds. Phytochemistry. 1987;26:1557–1566. [Google Scholar]
  36. Skriver K, Olsen FL, Rogers JC, Mundy J. Cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc Natl Acad Sci USA. 1991;88:7266–7270. doi: 10.1073/pnas.88.16.7266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A. The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell. 1999;11:431–443. doi: 10.1105/tpc.11.3.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Staswick PE. Storage proteins of vegetative plant tissues. Annu Rev Plant Physiol Plant Mol Biol. 1994;45:303–322. [Google Scholar]
  39. Thomas PS. Hybridization of denatured RNA transferred or dotted to nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
  40. Toki S. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol Biol Rep. 1997;15:16–21. [Google Scholar]
  41. Vierstra RD. Protein degradation in plants. Annu Rev Plant Physiol Plant Mol Biol. 1993;44:385–410. [Google Scholar]
  42. Von Heijne G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 1986;14:4683–4690. doi: 10.1093/nar/14.11.4683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Watanabe H, Abe K, Emori Y, Hosoyama H, Arai S. Molecular cloning and gibberellin-induced expression of multiple cysteine proteinases of rice seeds (oryzains) J Biol Chem. 1991;266:16897–16902. [PubMed] [Google Scholar]
  44. Xu F-X, Chye M-L. Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. Plant J. 1999;17:321–327. doi: 10.1046/j.1365-313x.1999.00370.x. [DOI] [PubMed] [Google Scholar]
  45. Ye Z-H, Varner JE. Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans. Plant Mol Biol. 1996;30:1233–1246. doi: 10.1007/BF00019555. [DOI] [PubMed] [Google Scholar]
  46. Youngdahl LJ, Pacheco R, Street JJ, Vlek PLG. The kinetics of ammonium and nitrate uptake by young rice plants. Plant Soil. 1982;69:225–232. [Google Scholar]
  47. Yu S-M, Kuo Y-H, Sheu G, Sheu Y-J, Liu L-F. Metabolic derepression of α-amylase gene expression in suspension-cultured cells of rice. J Biol Chem. 1991;266:21131–21137. [PubMed] [Google Scholar]
  48. Yu S-M, Lee Y-J, Fang S-C, Chan M-T, Hwa S-F, Liu L-F. Sugars act as signal molecules and osmotica to regulate the expression of α-amylase genes and metabolic activities in germinating cereal grains. Plant Mol Biol. 1996;30:1277–1289. doi: 10.1007/BF00019558. [DOI] [PubMed] [Google Scholar]
  49. Zhang NY, Jones BL. Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley. Planta. 1996;199:565–572. doi: 10.1007/BF00195188. [DOI] [PubMed] [Google Scholar]
  50. Zheng A, Hayashimoto A, Li Z, Mruai N. Hygromycin resistance gene cassettes for vector construction and selection of transformed rice protoplasts. Plant Physiol. 1991;97:832–835. doi: 10.1104/pp.97.2.832. [DOI] [PMC free article] [PubMed] [Google Scholar]

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