Ru-Huei Fu
1,2,∗, Ai-Yu Wang
1,2, Yu-Chi Wang
3& Hsien-Yi Sung
1,21Institute of Agricultural Chemistry,2Institute of Microbiology & Biochemistry, National Taiwan University, No. 1,
Sec. 4, Roosevelt Road, Taipei 106, Taiwan
3Department of Chemical Engineering, Cincinnati University, OH 45221, USA
∗Author for correspondence (Fax:+886-2-25791299; E-mail: f85623601@ms86.ntu.edu.tw)
Received 13 May 2003; Revisions requested 20 May 2003; Revisions received 27 June 2003; Accepted 3 July 2003
Key words: β-D-fructofuranosidase, cDNA, heterologously expression, Oryza sativa
Abstract
A vacuolar type β-D-fructofuranosidase (Osβfruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type β-D-fructofuranosidase of maize (Ivr1). Osβfruct3 exists as a single copy per genome. Northern analyses showed that Osβfruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Osβfruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type β-D-fructofuranosidase.
Introduction
β-D-Fructofuranosidases (EC 3.2.1.26) constitute a family of enzymes that hydrolyze sucrose into gluc-ose and fructgluc-ose (Tymowska-Lalanne & Kreis 1998, Sturm 1999). Several types of β-D-fructofuranosidase from rice (Oryza sativa) have been purified and char-acterized (Lin & Sung 1993, Sung & Huang 1994, Lin et al. 1999, Hsiao et al. 2002). However, information about gene expression and regulation of the rice
β-D-fructofuranosidase family is limited. In a previous paper, we have reported the cloning of a vacuolar type β-D-fructofuranosidase cDNA (Osβfruct2) from rice
and its expression in Pichia pastoris (Fu et al. 2003). In this present study, we describe the cDNA cloning, Southern and Northern analyses of another vacuolar β-D-fructofuranosidase gene (Osβfruct3) in etiolated rice seedlings. Osβfruct3 encodes a functional β-D -fructofuranosidase as demonstrated by the expression of active enzyme in Pichia pastoris.
Materials and methods
Preparation and screening of the cDNA library Total RNA was extracted from shoots of etiol-ated rice seedlings (germinetiol-ated for 8 d, Oryza sativa L. cv. Tainong no. 67) using the guan-idinium· HCl/thiocyanate/phenol/chloroform extrac-tion method. Polyadenylated RNA was isolated using Poly ATtract mRNA Isolation Systems (Promega), and RT-PCR was performed as the cDNA synthesis kit (Gibco BRL) with degenerate primers S1 (5
-AAAAACTGGATGAACGATCCTAATGGT-3) and
AS3 (5
-TCTTCCACCTTGAGCAAAGCTTTCAAC-3) derived from GenBank according to conserved sequences of vacuolar type β-D-fructofuranosidase of mung bean (Arai et al. 1992). The 1.41 kb frag-ment was used to screen a cDNA library constructed from the same etiolated rice seedlings RNA as used a λZAP-cDNA Synthesis Kit (Stratagene) using a Ran-dom Prime Labelling System (Amersham Pharmacia Biotech). A positive clone of Osβfruct3 was isolated and sequenced. In order to obtain the sequence of the 5region of Osβfruct3 cDNA, an amplification of the
1526
Fig. 1. Entire nucleotide sequence and deduced amino acid sequence of Osβfruct3. A putative signal peptide and N -terminal extension in the 5-coding region are underlined. Three conserved regions are marked by shadow boxes. Four potential N -glycosylation sites are indicated by double underlines. GenBank accession number of this nucleotide sequence is AY037871.
5ends (5RACE, Gibco BRL) was performed using a commercial dC-tailed cDNA from etiolated rice seed-lings as a template and the following primers: SP1 (5 -GGTGGAGTCGGAGCCCTCGTCGGTGGCCACG TC-3) and SP2 (5-GCGCTTGTCGTCCACCTCGT TCTCCTTGAAG-3). The deduced amino acids were analyzed for the putative targeting signal, isoelectric point and glycosylation sites using the Expert Protein Analysis System of Swiss Institute of Bioinformatics. The BLAST databanks were screened for similarity to other β-D-fructofuranosidase sequences.
Southern and Northern blot analyses
For Southern blot analysis, genomic DNA was pre-pared from the etiolated rice seedlings using the method of CTAB. Genomic DNA, approx. 10 µg, was
separately digested by EcoRI, NheI and SpeI, size-fractionated on 0.7% agarose gel and blotted onto nylon membrane (Millipore). The membrane was hy-bridized with PCR-labelled probes. A 265 bp PCR fragment of the 3 non-coding region of Osβfruct3 was prepared using a primer set of IT3-S: 5-TC
AGTCGATCAGCTAACCACTTACTGCGAA-3 and
IT3-AS: 5-ACTCACACACACAACTGGATC-3. Hy-bridization was carried out for 24 h at 42◦C. The blots were washed with 2× SSPE (3 M NaCl, 0.2 M sodium phosphate, 0.02 M EDTA, pH 7.4) plus 0.1% SDS at room temperature for 10 min, twice with 1× SSPE plus 0.1% SDS at 55◦C for 15 min, twice with 0.1× SSPE plus 0.1% SDS at 55◦C for 15 min and then exposed to X-ray film. For Northern blot analyses, total RNA was extracted by TRIzol reagent (Gibco
Fig. 2. Alignment of amino acid sequence of Osβfruct3 with those of homologous vacuolar type β-D-fructofuranosidases. The β-D-fructofuranosidase sequences (abbreviated name and accession number in brackets) are from rice (Osβfruct2: AY037870), mung bean (AI: D10265), Arabidopsis (Atβfruct3: X99111), carrot (sI: X75352) and maize (Ivr1: U16123).
1528
Fig. 3. Genomic DNA Southern blot analysis of etiolated rice seed-lings DNA using probes specific to the Osβfruct3. The sizes of molecular mass markers are indicated at the margin.
BRL), electrophoresed through agarose gels contain-ing formaldehyde. Transfer, hybridization, detection conditions were as described for genomic Southern blots. The amount of Osβfruct3 mRNA was quanti-fied using a Bioimaging analyzer (BAS1000, Fuji Co., Japan).
Expression of recombinant protein in yeast
Osβfruct3 was expressed in the methylotrophic yeast Pichia pastoris X-33 (EasySelect Pichia Expression Kit, Invitrogen), with the secretory expression vec-tor pPICZαA. The DNA sequence, corresponding to putative mature protein regions of Osβfruct3 between nucleotides 433 and 2148, was amplified by PCR and translationally fused behind the α-factor signal se-quence of pPICZαA. The PmeI-linearized vector with insert was transformed by EasyComp Kit (Invitrogen) into P. pastoris, and transformants were selected on YPDS-Zeocin agar plates (1% yeast extract, 2% pep-tone, 2%D-glucose, 1 M sorbitol, 2% agar, 100 µg Zeocin ml−1). A BMGY medium [1% (w/v) yeast ex-tract, 2% (w/v) peptone, 0.1 M potassium phosphate, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin and 1% (v/v) glycerol] was in-oculated with freshly prepared positive colonies and incubated for 48 h at 30 ◦C. The cells were collec-ted by centrifugation, transferred to a BMMY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 0.1 M
po-tassium phosphate, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin and 5% (v/v) methanol] and incubated at 28◦C under aerobic conditions. Every day, 100 µl methanol was added to 100 ml culture medium. Samples, 5 ml, were collected for up to 156 h.
Purification of recombinant protein
A nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Qiagen) was pre-equilibrated with 50 mM sodium phosphate buffer, pH 8, containing 300 mM NaCl and 5 mM imidazole. The yeast transformants were removed by centrifugation and the concentrated super-natant (Ultrafree15, Millipore) was loaded on to the column and washed with 50 mM sodium phosphate buffer, pH 8, containing 300 mM NaCl and 20 mM imidazole. Finally, the column was eluted with 50 mM sodium phosphate buffer, pH 8, containing 300 mM NaCl and 250 mM imidazole. Fractions containing acidic β-D-fructofuranosidase activity (method to see legend of Figure 5) were pooled and concentrated.
Results and discussion
Molecular characterization of vacuolar type β-D-fructofuranosidase from rice
A new vacuolar type β-D-fructofuranosidase isozyme cDNA (Osβfruct3, GeneBank accession number: AY037871) has been cloned here from rice. The cDNA fragment contains 2481 nucleotides with a ter-mination codon TGA and putative polyadenylation signal (AATAAT) (Figure 1). The cDNA encodes 688 amino acids with calculated relative molecular mass of 76 kDa and isoelectric point of 5. The protein contains all the elements characteristic of vacuolar type β-D -fructofuranosidase, including the β-fructosidase motif ‘NDPNG’ and the putative active site ‘WECVD’. The protein also contains four potential N-linked glyc-osylation sites (Figure 1). The N-terminus region, like other vacuolar type β-D-fructofuranosidase, contains signal peptide and N-terminal extension of 68 and 53 residues, respectively (Figure 1). The mature pro-tein have calculated molecular mass of 63 kDa and calculated pI value of 5.1.
Previously, we already reported one vacuolar type β-D-fructofuranosidase cDNA (Osβfruct2, GeneBank accession number: AY037870) from the same spe-cies (Fu et al. 2003). Compared to the previous
Fig. 4. Northern analyses of etiolated rice seedlings RNA using probes specific to the Osβfruct3 and 18S rRNA. (A) Tissue and stage specific expression. Total RNA from leaves, stems and roots of 8-d-old etiolated seedlings, 8-d-old green seedlings and mature plant of rice. (B) Stress specific expression. 8-d-old etiolated rice seedlings were exposed separately to stresses for 2 d. Total RNA from shoots of cold (exposed to 10◦C), heat (exposed to 40◦C), flooding (whole plants were submerged in distilled water), drought (dehydrated until losing 30∼40% of the fresh weight) and wounding (excised seedlings tip) treatments. (C) Sugars feeding experiments. Shoots were detached from etiolated rice seedlings (shoots were preadapted to the dark and soaked in distilled water for 5–6 h) and placed into a dish with a feeding solution (sucrose, glucose, fructose, sorbitol or mannitol) for 48 h.
79% identity at nucleotide level and 72% identit-ies at protein level. An alignment of the deduced amino acid sequences with various other vacuolar
β-D-fructofuranosidase from plants is shown in Figure 2. Osβfruct3 has a high similarity to the vacuolar β-D -fructofuranosidase of mung bean, Arabidopsis, carrot and maize with 60%, 58%, 63% and 58% identities, respectively.
Southern analysis
Total genomic DNA from etiolated rice seedlings di-gested with EcoRI, NheI and SpeI restriction enzymes, respectively, was hybridized with the Osβfruct3 probes (Figure 3). Only one hybridizing band was ob-served for Osβfruct3 showing that the gene is present in one copy per haploid genome.
Northern blot analyses
Osβfruct3 was highly expressed in leaves, stems, and roots of etiolated seedlings, green seedlings and rice plants. This contrasts with no expression in the grains and suspension cells. Osβfruct3 was also up-regulated by exposure of the plants to light (Figure 4A). For the stress treatments, Osβfruct3 transcription was in-creased in cold stress, flooding, drought and wounding (Figure 4B). For the feeding experiments, Osβfruct3 was strongly up-regulated by sucrose (<200 mM) and glucose (100 mM), but fructose, mannitol and sorbitol had no effect on expression (Figure 4C). Northern ana-lyses showed that Osβfruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars.
Expression and properties of the recombinant OsβFRUCT3
To confirm that Osβfruct3 encodes a vacuolar type β-D-fructofuranosidase, the Osβfruct3 cDNA was ex-pressed in the Pichia pastoris. A clear sucrose
hydro-1530
Fig. 5. Time-course of OsβFRUCT3 expression in P. pastoris trans-formed with pPICZαA vector system. (), Cell dry weight; (), vacuolar β-D-fructofuranosidase activity in medium (P. pastoris was transformed with Osβfruct3); (), vacuolar β-D-fructofuranosidase activity in medium (P. pastoris was only transformed with pPICZαA); (), intracellular vacuolar β-D-fructofuranosidase activity (P. pastoris was transformed with Osβfruct3). Protein concentration was determined by the protein-dye binding method. The enzyme activity was assayed in a 0.36 ml mixture of 0.2 M sucrose in 100 mM sodium acetate (pH 5) for vacuolar β-D-fructofuranosidase, at 37◦C for 10 min and the amount of reducing sugar was measured by the Somogyi–Nelson method. One enzyme unit was defined as the amount of enzyme that catalyzed the production of 1 µmol reducing sugar per min at 37◦C and at the optimum pH.
lysis activity was only detected around the transform-ant with the Osβfruct3 expression vector (Figure 5). This result suggests that the active recombinant en-zyme was expressed in the yeast and secreted from cells. To characterize OsβFRUCT3, the recombin-ant enzymes were purified and subjected to SDS-PAGE and showed a single protein band of about 67 kDa that matched the calculated molecular mass of OsβFRUCT3 (Figure 6). Recombinant enzymes were incubated for 10 min at various temperatures or in different pH. Activity of the recombinant enzymes was optimal at 30◦C and in pH 4.5 (data not shown). The biochemical properties detailed above are similar to those of vacuolar β-D-fructofuranosidase purified from rice (Lin & Sung 1993) and various other plants (Fu et al. 2002).
Acknowledgement
This work was supported in part by the National Science Council of the Republic of China.
Fig. 6. Production of OsβFRUCT3 in P. pastoris. The figure shows the results of 10% SDS-PAGE analysis of the produc-tion of OsβFRUCT3 throughout the time course of the fer-mentation. Silver staining was used to visualize the protein. Lane M, low-molecular-mass markers; lane 1, purification of nickel-nitrilotriacetic acid (Ni-NTA) agarose column; lanes 2–9, cell-free medium (10 µl) 0–144 h after 0.5% methanol induction, respectively. The arrow indicates the position of the 67 kDa of OsβFRUCT3.
References
Arai M, Mori H, Imaseki H (1992) Cloning and sequence for an in-tracellular acid invertase from etiolated hypocotyls of mung bean and expression of the gene during growth of seedlings. Plant Cell Physiol. 33: 245–252.
Fu RH, Wang YL, Sung HY (2002) Review: biochemical and mo-lecular biological studies in plant invertase. Food Sci. Agric. Chem. 4: 1–7.
Fu RH, Wang YL, Sung HY (2003) Cloning, characterization and functional expression of a new β-D-fructofuranosidase (Osβfruct2) cDNA from Oryza sativa. Biotechnol. Lett. 25: 455–459.
Hsiao CC, Fu RH, Sung HY (2002) A novel bound form of plant invertase in rice suspension cells. Bot. Bull. Acad. Sin. 43: 115– 122.
Lin CL, Lin HC, Wang AY, Sung HY (1999) Purification and char-acterization of an alkaline invertase from shoots of etiolated rice seedlings. New Phytol. 142: 427–434.
Lin SS, Sung HY (1993) Partial purification and characterization of soluble acid invertases from rice (Oryza sativa) leaves. Biochem. Mol. Biol. Int. 31: 945–953.
Sturm A (1999) Invertases. Primary structures, functions, and roles in plant development and sucrose partitioning. Plant Physiol.
121: 1–7.
Sung HY, Huang WC (1994) Purification and characterization of cell-wall-bound invertase from rice (Oryza sativa) grains. Biotechnol. Appl. Biochem. 19: 75–83.
Tymowska-Lalanne Z, Kreis M (1998) The plant invertase: physiology, biochemistry and molecular biology. In: Callow JA, ed. Advances in Botanical Research, Vol. 28. New York: Academic Press, pp. 71–117.
