Breeding Science 54 : 67-73 (2004)

Disruption of GSTZ1 by Large Genetic Alteration in glaberrima

Tokuji Tsuchiya and Ikuo Nakamura*

Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Japan

After the completion of the genome sequencing project Introduction of common ( L.), comparative genomic studies between rice and related species became impor- S-transferases (GSTs; EC 2.5.1.18) are tant to reveal the function of each gene. The rice ge- ubiquitous and abundant detoxifying in all the organ- nome contains two copies of the gene encoding zeta class isms, such as bacteria, fungi, animals and . Recently, glutathione S-transferase (GSTZ) that is reported to be GSTs have been classified into four different classes, the in the catabolic pathway of and phi, tau, theta and zeta, based on sequence simi- . Two GSTZ of O. sativa, OsGSTZ1 larity and gene structure (Dixon et al. 1998, Edward et al. and OsGSTZ2, display a tandem arrangement. Up- 2000). The phi and tau GST genes are plant-specific and com- stream OsGSTZ1 gene is constitutively expressed, pose large multi-gene families, whereas the theta and zeta whereas the downstream OsGSTZ2 gene is inducible by GST genes have a few copies. The zeta class GST (GSTZ) stresses. We analyzed the expression of the GSTZ gene genes are present as one or two copies in every plant genome in the African cultivated species O. glaberrima and wild studied, such as A. thaliana, maize, soybean, carnation and species O. longistaminata by using RT-PCR. The results rice (Meyer et al. 1991, Raghothama et al. 1991, McGonigle showed that both GSTZ1 and GSTZ2 genes were ex- et al. 2000, Dixon et al. 2002). Since the plant GSTZ genes pressed in O. longistaminata, whereas only the cDNA are highly homologous to those of mammals and fungi fragment of downstream GSTZ2 was detected in (Board et al. 1997, Blackburn et al. 1998, Subramaniam O. glaberrima. Thus, the genomic sequence of the GSTZ1 et al. 1999), GSTZ genes have been considered to encode locus of O. glaberrima was determined by PCR genomic common essential proteins in all eukaryotes (Hayes and walking. Sequence comparison between O. sativa and Mclellan 1999). Actually, human and fungal GSTZ proteins O. glaberrima revealed that the genomic sequence up- occur as maleylacetoacetate isomerase (MAAI) in phenyl- stream from the eighth intron of the OgGSTZ1 gene was alanine and tyrosine metabolic pathways (Fernandez-Canon highly homologous, in inverted orientation, to a BAC and Penalva 1998). Thom et al. (2001) showed that the clone, over 1-Mb apart from the OsGSTZ1 gene in GSTZ protein in Arabidopsis exhibited a similar activity to O. sativa. This result suggested that the OgGSTZ1 gene that of MAAI. The function of MAAI is important because was disrupted by a large rearrangement or inversion. MAAI might be involved in the nitrogen metabolism in The genetic alteration was also observed in several lines plants. of O. glaberrima and its ancestral wild species O. barthii. We have characterized the structure and expression of The loss of the OgGSTZ1 gene in O. glaberrima is im- the GSTZ genes in rice (Oryza sativa L.). Rice contains two portant, as the expression of the gene is 12-fold higher copies of the GSTZ gene, OsGSTZ1 and OsGSTZ2, in a tan- than that of OsGSTZ2 in O. sativa and since O. sativa dem arrangement orientation on the short arm of chromo- and O. glaberrima differ in several such as seed yield some 12. The upstream OsGSTZ1 and downstream OsGSTZ2 and annual/perennial habitat that might be related to genes display a complex structure with ten and nine , the translocation of nitrogen metabolites from leaves to respectively, in spite of encoding a relatively short coding seeds. sequence (ca. 700bp). The amino acid sequences deduced from the OsGSTZ1 and OsGSTZ2 genes were highly homol- Key Words: , glutathione S-transferase, ogous (77.9 %). Real-Time RT-PCR revealed that OsGSTZ1 maleylacetoacetate isomerase, tyrosine was expressed at a level ca. 12-fold higher than that of metabolism, comparative genomics, PCR OsGSTZ2 in the leaves of rice plants. OsGSTZ1 was express- genomic walking. ed in a relatively constitutive manner under various stress conditions. In contrast, the expression of the OsGSTZ2 gene was strongly enhanced (30-fold) by treatment with jasmonate (Tsuchiya et al., data not shown). There are two cultivated species of rice, Oryza sativa and O. glaberrima Steud. The former species is common rice widely grown in tropical and temperate world wide and Communicated by K. Kadowaki differentiated into various subspecies, such as indica and Received October 14, 2003. Accepted November 6, 2003. japonica, while the latter is a monomorphic species endemic *Corresponding author (e-mail: [email protected]) to West (Morishima et al. 1984). Oryza sativa and 68 Tsuchiya and Nakamura

O. glaberrima originated independently from wild relatives, from both ends using universal primers. the O. rufopogon-nivara complex in Southeast Asia and O. barthii in , respectively. As F1s between Sequencing of OgGSTZ locus O. sativa and O. glaberrima are sterile, the genome symbol was Two different methods of PCR genomic walking were designated as AA for O. sativa and AgAg for O. glaberrima. employed to obtain sequence information around the However, the chromosomal pairing in their hybrid was nor- OgGSTZ locus in O. glaberrima. About 1-kb fragment up- mal (Morinaga and Kuriyama 1957). The chromosomal or- stream of OgGSTZ2 was amplified by TAIL-PCR (Liu et al. ganization and location of six different loci (wx, A, Se-1, Rc, 1995) using random RP primer and P5, P6 and P7 as gene 5S and 45S rDNA) are comparable between the two species specific primers (Table 1 and Fig. 2). The sequence further (Sano 1988, Ohmido and Fukui 1995). Oka (1988) pointed upstream (ca. 2-kb) was isolated by Anchor-PCR (Troutt et out that O. glaberrima exhibited stress tolerance to drought, al. 1992). Briefly, gDNA of the O. glaberrima line CG14 diseases and weeds, while O. sativa showed a high seed was digested by BglII and ligated with an anchor (AD-F, yield of seeds and was susceptible to various kinds of biotic AD-R, Table 1). PCR reactions were performed twice using and abiotic stresses. two OgGSTZ2 specific primers (P11 and P12) and adapter After the completion of the rice genome sequence primers (AP1 and AP2). The gDNA and cDNA sequences of project, the comparison of genomic sequences between rice the OgGSTZ2 gene were also amplified using P8, P9 and P10 and related species may enable to study the function of the primers. The amplified fragments were directly sequenced genes. We examined the expression of the OgGSTZ genes in or cloned into pCR2.1 plasmid (Invitrogen). Homology O. glaberrima because O. sativa and O. glaberrima display search was carried out through NCBI Blast server (Altschul differences in seed production and annual-perennial habitat. et al. 1997). That may possibly be related to the translocation of nitrogen metabolites. In this paper, it was observed that the OgGSTZ1 PCR amplification of the altered region gene of O. glaberrima was disrupted by a large genetic re- A pair of primers (P13 and P14) was designed to ampli- arrangement or inversion over 1-Mb of the chromosomal fy the fragment containing the genetic alteration found in fragment. Comparative genetics of O. glaberrima may also O. glaberrima. PCR reactions were executed using DNA give the opportunity to reveal the physiological function of templates extracted from five lines of O. glaberrima and three the GSTZ1 gene. lines of O. barthii. The amplified products were subjected to agarose gel electrophoresis. Materials and Methods Southern blot analysis Plant materials A DIG-labeled probe for OgGSTZ2 cDNA was pre- Oryza sativa ‘’, O. glaberrima (lines CG14, pared using P9 and P10 primers and a PCR DIG probe syn- C0028, C0039, C0416, C7290 and C7293), O. barthii (lines thesis kit (Roche). Aliquots of 5 µg gDNA were digested by W0652, W0720 and W0747) and O. longistaminata (line eight appropriate restriction endonucleases that did not W1413) were obtained from Iwate Agricultural Research Center and the National Institute of Genetics, Japan. These Table 1. List of primers used in this study rice plants were grown in a greenhouse and leaves at the adult stage were harvested. Genomic DNAs (hereafter re- Name Sequence (5′→3′) ferred to as gDNA(s)) and total RNAs were isolated from the P1 AGCATTTCAAGCTGCTGTC leaves by the CTAB (Rogers and Bendich 1988) and phenol/ P2 TATGGCACTGGAGACTACTCA SDS (Shirzadegan et al. 1991) methods, respectively. Total P3 CGTCAAAGCCAATCCTGTA CTCCATCTACTAATGCTGGTATG RNA (2 µg) was reverse-transcribed using a ReverTra Ace P4 AGCTAAAGCAGTAAGAGTGTTGACT Kit (Toyobo) to synthesize cDNA, according to the manu- P5 P6 AGCTCAGTGAAGCCAAAGAATCAA facturer’s instructions. P7 CTCCAATTGCTCAAGAAATGCTTAC P8 AACTAAACGTGGCATTTATTCACTTACATA PCR amplification of gDNA and cDNA for GSTZ genes P9 AGGTACTGGAGTACTGGATGGATAAT PCR was performed to amplify gDNA and cDNA of the P10 ACCTTTTCAGGACAGCATCTACTC GSTZ gene in O. glaberrima (CG14) and O. longistaminata P11 CATCTGGTTGGTTTTGAGAATGTA (W1413). Two pairs of specific primers, P1-P2 and P3-P4 P12 AGTATTCAATAGGATTCTCTTGTCACTTAGA (Fig. 2 and Table 1), were designed based on the cDNA se- P13 GAAGGTGCATCTGGCTGATTCTTT quences of OsGSTZ1 (Genbank AF309381) and OsGSTZ2 P14 GCGTCGTCAAAGCCAATCCTGTA (Genbank AF402792), respectively. The amplified products RP GTNCGA(G/C)(A/T)CANA(A/T)GTT were fractionated through a 1 % agarose gel and visualized AD-F CGCAGGCTGGCAGTCTCTTTAGGGTTACACGATTGCTT by staining with ethidium bromide under a UV trans- AD-R TCAAGCAATCGTGT illuminator. Furthermore, the amplified fragments were AP1 CGCAGGCTGGCAGTCTCTTTAG ATGCGGCCGCTCTCTTTAGGGTTACACGATTGCTT cloned into a pCR2.1 plasmid (Invitrogen) and sequenced AP2 Disruption of GSTZ1 gene by genetic alteration in Oryza glaberrima 69 digest the inside part of the OgGSTZ2 gene. The digested ever, we detected a large genetic alteration at the eighth intron DNAs were fractionated by electrophoresis through a 0.8 % of the OgGSTZ1 gene because the 2-kb upstream sequence agarose gel and blotted onto a positively charged nylon was highly homologous (95.9 %) to that of the BAC clone membrane Biodyne B (PALL). Pre-hybridization and hy- OSJNBa0029N15 in inverted orientation. The clone bridization were performed at 41°C in DIG Easy Hyb OSJNBa0029N15 was located at more than 1-Mb apart from (Roche) using the DIG-labeled OgGSTZ2 probe. The mem- the OsGSTZ1 gene in O. sativa. branes were washed twice in 2 × SSC, 0.1 % SDS for 5 min- The gDNA and cDNA sequences (AB121667, utes and then washing was performed again twice for 15 AB121668) of the OgGSTZ2 gene were also determined by minutes in 0.5 × SSC, 0.1 % SDS. The signals were detected using P8, P9 and P10 primers. Sequence analysis revealed by the chemi-luminescence method using CDP-star sub- the presence of the ninth and tenth exons of the OgGSTZ1 strate (DIG Luminescent Detection Kit), according to the gene in O. glaberrima. The deduced amino acid sequences manufacturer’s instructions (Roche). showed a 100 % and 99.5 % homology for the ninth be- tween OsGSTZ1 and OgGSTZ1 (Fig. 4) and for all the exons Results between OsGSTZ2 and OgGSTZ2 (data not shown), respec- tively. Southern blot analysis indicated that the GSTZ gene The two GSTZ genes were located in a tandem arrange- was present as a single copy in O. glaberrima, using eight ment on the short arm of 12 in O. sativa (Fig. different kinds of restriction enzymes (Fig. 5). This result 1A). The upstream OsGSTZ1 and downstream OsGSTZ2 also suggested that the OgGSTZ1 gene was lost in the ge- genes displayed a very complex structure with ten and nine nome of O. glaberrima. exons, respectively, although these genes encoded for rela- A pair of primers (P13 and P14) was designed to con- tively small proteins (ca. 25 kDa). Two pairs of primers (P1- firm the genetic alteration. When genomic DNAs from five P2 or P3-P4) specific to either the GSTZ1 or GSTZ2 genes of O. glaberrima and three O. barthii lines were used as PCR O. sativa were designed, respectively (Table 1). It was as- template, the predicted products with the same sized (719 sumed that the P1-P2 primers amplified 411 bp and 273 bp bp) were amplified in all the tested lines (Fig. 6). This result fragments derived from the gDNA and cDNA of OsGSTZ1, showed that the large genetic alteration at the OgGSTZ1 respectively, while the P3-P4 primers produced 1,093 bp locus was not specific to line CG14 and also occurred in and 171 bp fragments for OsGSSTZ2 (Fig. 1A). PCR ampli- O. glaberrima and its wild ancestor O. barthii. Figure 7 shows fication using these primers revealed that both GSTZ1 a schematic representation of the genomic structures at the and GSTZ2 genes were present and expressed in the GSTZ locus in O. sativa. The sequence upstream from the O. longistaminata line W1413 as well as in the O. sativa eight intron of the OgGSTZ1 gene was highly homologous, (Fig. 1B). In O. glaberrima line CG14, although the same in inverted orientation, to the BAC clone OSJNBa0029N15 amplified products were found only in the GSTZ2 genes, un- on the short arm of chromosome 12. The altered region of expectedly larger products were detected for both gDNA O. glaberrima corresponded to nine BAC ordered clones be- and cDNA of GSTZ1. The larger fragment from cDNA was tween OSJNBa0029N15 and OSJNBb0071I17 in O. sativa. directly sequenced and derived from another isomerase (cyclophilin) gene and not from the MAA isomerase (GSTZ) Discussion gene. These results suggested that the GSTZ1 gene might Glutathione S-transferases (GSTs) are generally con- have been lost in the genome of O. glaberrima and, there- sidered to be the enzymes that detoxify intra- and extra- fore, the genomic sequence of the OgGSTZ locus was ana- cellular zenobiotics. Recently, human and Arabidopsis GSTZ lyzed. About 1-kb fragment upstream from OgGSTZ2 was proteins have been identified as an isomerase for the cataly- amplified by TAIL-PCR using random RP primer and P5, sis of cis-trans conversion of MAA, an intermediate product P6 and P7 as gene specific primers (Table 1 and Fig. 2). in the phenylalanine and tyrosine catabolic pathway Since TAIL-PCR was not successed, Anchor-PCR was em- (Fernandez-Canon and Penalva 1998, Dixon et al. 2000). ployed to amplify the region further upstream. Genomic The function of MAA isomerase suggests that GSTZ pro- DNA of O. glaberrima was digested by BglII and ligated teins are involved in the re-utilization and translocation of with an adapter (AD-F, AD-R, Table 1). Nested PCR reac- nitrogen metabolites from source to sink in plants. tions were carried out using two OgGSTZ2-specific primers We were interested in the expression of the GSTZ gene (P11 and P12) and adapter primers (AP1 and AP2). We were in O. glaberrima, African annual cultigen, because although eventually able to amplify the ca. 2-kb upstream fragment O. glaberrima is a close relative of O. sativa, the two culti- (Fig. 3). The amplified fragments were sequenced and the gens differ in seed yield and annual/perennial habitat that determined sequence (AB121668) was aligned with that of might be related to the translocation of nitrogen metabolites O. sativa provided by the Rice Genome Project (Fig. 4). Se- from vegetative to reproductive tissues. At the beginning, quence comparison revealed the absence of PCR product for PCR analysis using specific primers to either OsGSTZ1 or the gDNA and cDNA of OgGSTZ1 (Fig. 1) due to the 17-bp OsGSTZ2 showed that the amplified products for both insertion at the P2 primer position in O. glaberrima. How- GSTZ1 and GSTZ2 genes were present in line W1413 of 70 Tsuchiya and Nakamura

Fig. 1. A: Genomic structure of tandem-arranged OsGSTZ1 and OsGSTZ2 genes in O. sativa. Positions of exons (filled box), initiation (Init), stop (Stop) and polyadenylation (p(A)) of each GSTZ gene are indicated. Two pairs of PCR primers (P1-P2, P3-P4) used in Fig. 1B amplified the fragments with respec- tive sizes from genomic (g) DNA and cDNA of the two GSTZ genes. B: PCR products specific to either GSTZ1 or GSTZ2 gene in O. sativa, O. longistaminata and O. glaberrima. DNA fragments were amplified using GSTZ1 (P1-P2) or GSTZ2 (P3-P4) gene-specific primers shown in Fig. 1A and PCR templates of gDNA or cDNA were prepared from O. sativa (lanes 1, 4, 7, 10), O. longistaminata (lanes 2, 5, 8, 11) and O. glaberrima (lanes 3, 6, 9, 12). M: φX174/HaeIII molecular weight marker.

Fig. 2. Schematic representation showing the position and direction of PCR primers. PCR primers, listed in the table, were used for sequencing and genomic walking of GSTZ1 and GSTZ2 genes in O. sativa and O. glaberrima. Disruption of GSTZ1 gene by genetic alteration in Oryza glaberrima 71

Fig. 3. PCR product containing the upstream fragment from the OgGSTZ1 gene in O. glaberrima. Arrow indicates the ampli- fied fragment (ca. 2-kb) isolated using P11 and P12 primers by Anchor-PCR strategy. A: Amplified product by second round Anchor-PCR, M: λDNA/HindIII molecular weight marker.

Fig. 4. Alignment of genomic sequence around GSTZ1 gene between O. sativa and O. glaberrima. Genomic sequences of GSTZ1 region in O. glaberrima (gla) line CG14 and O. sativa (sat) ‘Sasanishiki’ were aligned and asterisks indicate identical nu- cleic acids. A shaded box indicates the junction of the genomic alteration in O. glaberrima. The donor and acceptor sites of the intron in the GSTZ1 gene are denoted by hollow characters and the stop codon is boxed. Primers (P1, P2, P13, P14) were used in this study and the deduced amino acid sequence of the ninth exon of GSTZ1 is shown in one letter.

O. longistaminata, African perennial wild species, while only the GSTZ2 gene was detected in line CG14 of O. glaberrima (Fig. 1). As those findings indicated the presence of some ge- netic alterations in the OgGSTZ1 gene, attempts were made to isolate the upstream fragment from the OgGSTZ2 gene Fig. 5. Southern blot analysis of O. glaberrima using the because the two GSTZ genes of O. sativa were arranged in OgGSTZ2 cDNA fragment as a probe. Genomic DNAs tandem with a short intergenic sequence (Tsuchiya et al., were digested by SalI, PstI, KpnI, XhoI, BamHI, BglII, data not shown). About 1-kb and additional 2-kb fragments SpeI and SphI (lanes 1-8), and were absent in the upstream from the OgGSTZ2 gene were amplified by TAIL- OgGSTZ2 gene. PCR and Anchor-PCR (Fig. 3), respectively. Sequence anal- 72 Tsuchiya and Nakamura ysis of the cloned fragments suggested that a large alteration genetic alteration had occurred probably when O. barthii over 1-Mb was present at the eighth intron of the OgGSTZ1 evolved because the deduced amino acid sequence encoded gene in the O. glaberrima line CG14 (Fig. 4). The loss of the by the ninth exon of OgGSTZ1 gene was completely identi- OgGSTZ1 gene was also comfirmed by Southern blot analy- cal (with three non-sense substitutions) with that of the sis because the GSTZ gene was detected as a single copy OsGSTZ1 gene (Fig. 4). (Fig. 5). PCR amplification which was performed to confirm The sequence upstream (ca. 2 kb) from the eight intron the alteration of the region with P13 and P14 primers indi- of the OgGSTZ1 gene was highly homologous (95.9 %), in cated that the alteration occurred not only in O. glaberrima inverted orientation, to the BAC clone OSJNBa0029N15 on but also in the wild relatives of O. barthii (Fig. 6). These re- the short arm of chromome 12. The BAC clone sults suggested that the OgGSTZ1 gene had been lost from OSJNBa0029N15 was located over 1-Mb apart from the the genome during the genetic alteration. Additionally, this OsGSTZ1 gene on the BAC clone OSJNBb0071I17 beyond

Fig. 6. PCR analysis to confirm the genomic alteration of the GSTZ1 gene in O. glaberrima and O. barthii. PCR amplification beyond the junction using P13 and P14 primers produced a fragment with expected size (arrow, 732 bp) that was present in five lines (lanes 1–5: C0028, C0039, C0416, C7290, C7293) of O. glaberrima and three lines (lanes 6–8: W0652, W0720, W0747) of O. barthii, and absent in ‘Sasanishiki’ (Os) of O. sativa. M: φx174/HaeIII molecular weight marker.

Fig. 7. Schematic representation of nine BAC ordered clones on the short arm of chromosome 12 in O. sativa. Positions of two GSTZ genes, two ESTs (R3375, C98313) and GAP are indi- cated. Arrows in box illustrate the positions and directions of two homologous fragments. OgGSTZ1 gene might be disrupted by a large genetic rearrangement or inversion over 1- Mb corresponding to nine BAC ordered clones between OSJNBa0029N15 and OSJNBb0071I17 in O. glaberrima. Disruption of GSTZ1 gene by genetic alteration in Oryza glaberrima 73 seven-ordered BAC clones in O. sativa. This suggested that S-transferases, enzymes with multiple functions in sickness the OgGSTZ1 gene of O. glaberrima was disrupted by a and in health. Trends Plant Sci. 5: 193-198. large rearrangement or inversion between OSJNBa0029N15 Fernandez-Canon, J.M. and M.A. Penalva (1998) Characterization of a (31.8 cM) and OSJNBb0071I17 (40.6 cM) (Fig. 7). fungal maleylacetoacetate isomerase gene and identification of It was remarkable to note that the GSTZ1 gene had been its human homologue. J. Biol. Chem. 273: 329-337. Hayes, J.D. and L.I. Mclellan (1999) Glutathione and glutathione- lost in O. glaberrima because the GSTZ1 gene was the main dependent enzymes represent a co-ordinately regulated defence constitutive gene expressed at a 12-fold higher level than the against . Free Radic. Res. 31: 273-300. GSTZ2 gene and the GSTZ2 gene was induced 30-fold by Liu, Y.-G. and R.F. Whittier (1995) Thermal asymmetric interlaced jasmonate in O. sativa (Tsuchiya et al., data not shown). 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