Isolation and Characterization of a Novel GRAS Gene That Regulates Meiosis-associated Gene Expression

Abstract

GRAS protein is a family of plant-specific proteins that plays a role in various developmental processes. Here we report a novel GRAS protein from lily, designated LlSCL (Lilium longiflorum Scarecrow-like), dominantly expressed at the premeiotic phase within anthers. The LlSCL protein has two highly basic regions, and transient expression analyses of dissected GFP-LlSCL fusion proteins showed that both basic regions are important for the nuclear localization. A series of transcriptional activation experiments of truncated LlSCL proteins fused to the yeast GAL4 DNA-binding domain clearly demonstrated that the amino terminus of the LlSCL protein has a strong activity of transcriptional activation in the yeast as well as in the plant cell. Further investigation on the effect of the LlSCL protein on the transcriptional activity of the meiosis-associated promoter revealed that in pollen mother cells of the lily, the activity of the meiosis-associated promoter is specifically enhanced by LlSCL protein co-expression. These results suggest that LlSCL is involved in transcriptional regulation during microsporogenesis within the lily anther. GRAS protein is a family of plant-specific proteins that plays a role in various developmental processes. Here we report a novel GRAS protein from lily, designated LlSCL (Lilium longiflorum Scarecrow-like), dominantly expressed at the premeiotic phase within anthers. The LlSCL protein has two highly basic regions, and transient expression analyses of dissected GFP-LlSCL fusion proteins showed that both basic regions are important for the nuclear localization. A series of transcriptional activation experiments of truncated LlSCL proteins fused to the yeast GAL4 DNA-binding domain clearly demonstrated that the amino terminus of the LlSCL protein has a strong activity of transcriptional activation in the yeast as well as in the plant cell. Further investigation on the effect of the LlSCL protein on the transcriptional activity of the meiosis-associated promoter revealed that in pollen mother cells of the lily, the activity of the meiosis-associated promoter is specifically enhanced by LlSCL protein co-expression. These results suggest that LlSCL is involved in transcriptional regulation during microsporogenesis within the lily anther. All sexually reproducing organisms have a specialized developmental pathway for gametogenesis, in which diploid cells undergo meiosis to produce haploid germ cells. In plant male gametogenesis, archesporial cells differentiate into pollen mother cells (PMCs), 1The abbreviations used are: PMC, pollen mother cell; UAS, upstream activating sequence; 5′-RACE, 5′-rapid amplification of cDNA ends; GFP, green fluorescent protein; CaMV, cauliflower mosaic virus; NLS, nuclear localization signal; BR, basic region; DB, DNA-binding domain; AD, activation domain; STAT, signal transducers and activators of transcription. and the PMCs give rise to the productions of tetrads of four haploid germ cells with two nuclear divisions. Subsequently, the haploid germ cells develop into mature pollen through the cytological and molecular events involved in the pollen developmental pathway. Meiosis organizes the transition from diploid to haploid and thus is one of the most complex events to occur during gametogenesis. The complexity of events suggests that many genes are tightly regulated to ensure each successful meiotic division. In yeast, characterization of temporal and spatial gene expression at meiosis has contributed toward a greater understanding of the mechanism of the meiotic gene (1Chu S. DeRisi J. Eisen M. Mulholland J. Botstein D. Brown P.O. Herskowitz I. Science. 1998; 282: 699-705Crossref PubMed Scopus (1539) Google Scholar). In higher eukaryotes, however, only a limited number of meiotic genes have been reported because of the lack of appropriate analytical techniques. The monocotyledonous plant Lilium longiflorum has been used for decades to study meiosis because of the accessibility of the male gametophyte (microsporocytes and pollen grains) and the synchronous development among sporogenous cells within an anther (2Erickson R.O. Am. J. Bot. 1948; 35: 729-739Crossref Google Scholar, 3Hotta Y. Tabata S. Stubbs L. Stern H. Cell. 1985; 40: 785-793Abstract Full Text PDF PubMed Scopus (24) Google Scholar). By using the subtractive method, Kobayashi et al. (4Kobayashi T. Kobayashi E. Sato S. Hotta Y. Miyajima N. Tanaka A. Tabata S. DNA Res. 1994; 1: 15-26Crossref PubMed Scopus (98) Google Scholar) isolated 18 cDNAs (LIM1–LIM18 genes), which are preferentially expressed during the premeiotic phase of microsporogenesis in L. longiflorum. LIM15 gene, which shows a similarity to the DNA recombination gene RecA, has been shown to be involved in meiosis in plants (5Terasawa M. Shinohara A. Hotta Y. Ogawa H. Ogawa T. Genes Dev. 1995; 9: 925-934Crossref PubMed Scopus (155) Google Scholar, 6Couteau F. Belzile F. Horlow C. Grandjean O. Vezon D. Doutriaux M.-P. Plant Cell. 1999; 11: 1623-1634Crossref PubMed Scopus (262) Google Scholar). These results suggest that genes induced at meiosis have a function associated with meiotic events. However, mechanisms involved in meiosis-specific gene expression are not necessarily clear, and factors that regulate gene expression specific to microsporogenesis have not been identified yet. In the view of gene expression at microsporogenesis, identification of the factors that regulate specific gene expression should provide important clues to elucidate mechanisms involved in meiotic events in higher plants. In an attempt to obtain information for genes expressed during microsporogenesis and meiosis, we previously carried out a large scale sequencing project using a lily zygotene cDNA library (7Morohashi K. Takase H. Hotta Y. Hiratsuka K. Plant Biotechnol. 2000; 17: 131-135Crossref Scopus (5) Google Scholar). One of the sequenced clones, M1125, has homology with a plant-specific putative transcription factor, Scarecrow (SCR). SCR is required for asymmetric cell division in an Arabidopsis root and encodes a novel putative transcription factor (8Di Laurenzio L. Wysocka-Diller J. Malamy J.E. Pysh L. Helariutta Y. Freshour G. Hahn M.G. Feldmann K.A. Benfey P.N. Cell. 1996; 86: 423-433Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). Recently, SCR-like genes were reported in various plant species such as maize and pea (9Lim J. Helariutta Y. Specht C.D. Jung J. Sims L. Bruce W.B. Diehn S. Benfey P.N. Plant Cell. 2000; 12: 1307-1318Crossref PubMed Scopus (85) Google Scholar, 10Sassa N. Matsushita Y. Nakamura T. Nyunoya H. Plant Cell Physiol. 2001; 42: 385-394Crossref PubMed Scopus (20) Google Scholar). SCR-like gene functions are not restricted to the asymmetric cell division. Although Repressor of Ga1–3 (RGA) and gibberellin-insensitive (GAI) genes show a similarity to SCR in their amino acid sequences, they play important roles in the gibberellin signal transduction of Arabidopsis but not in asymmetric cell division (11Silverstone A.L. Ciampaglio C.N. Sun T.-P. Plant Cell. 1998; 10: 155-169Crossref PubMed Scopus (612) Google Scholar, 12Peng J.P. Carol P. Richards D.E. King K.E. Cowling R.J. Murphy G.P. Harberd N.P. Genes Dev. 1997; 11: 3194-3205Crossref PubMed Scopus (915) Google Scholar). In addition, PAT1 protein, which shows a similarity to SCR protein, has been shown to be involved in the phytochrome A signal transduction of Arabidopsis (13Bolle C. Konez C. Chua N.-H. Genes Dev. 2000; 14: 1269-1278PubMed Google Scholar). In other species, various functions of SCR-like genes were reported; the Lateral suppressor (Ls) gene in tomato has crucial functions in the formation of lateral branches, and SLR1 of rice has been identified as an ortholog of GAI (14Schumacher K. Schmitt T. Rossberg M. Schmitz G. Theres K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 290-295Crossref PubMed Scopus (354) Google Scholar, 15Ikeda A. Ueguchi-Tanaka M. Sonoda Y. Kitano H. Koshioka M. Futsuhara Y. Matsuoka M. Yamaguchi J. Plant Cell. 2001; 13: 999-1010Crossref PubMed Scopus (615) Google Scholar). Pysh and co-workers (16Pysh L.D. Wysocka-Diller J.W. Camilleri C. Bouchez D. Benfey P.N. Plant J. 1999; 18: 111-119Crossref PubMed Scopus (527) Google Scholar) identified a number of Arabidopsis ESTs (expressed sequence tags) that showed a similarity to SCR amino acid sequence and designated them Scarecrow-like (SCL). They indicated that SCL genes comprised a novel gene family, referred to as the GRAS gene family. The GRAS gene products have conserved carboxyl termini, but the amino termini are structurally diverse. It has been suggested that the diversity of amino termini would be related to their functions, but no detailed studies on functional analysis have been carried out. Although the SCR product is predicted to be a transcriptional regulator on the basis of structural similarities to transcriptional regulatory proteins (8Di Laurenzio L. Wysocka-Diller J. Malamy J.E. Pysh L. Helariutta Y. Freshour G. Hahn M.G. Feldmann K.A. Benfey P.N. Cell. 1996; 86: 423-433Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar), direct evidence for the GRAS gene product as a transcriptional regulator has not been reported. It was shown that chimeric SLR1/OsGAI proteins fused to the yeast GAL4 DNA-binding domain enhanced UAS promoter activity (17Ogawa M. Kusano T. Katsumi M. Sano H. Gene. 2000; 245: 21-29Crossref PubMed Scopus (101) Google Scholar, 18Itoh H. Ueguchi-Tanaka M. Sato Y. Ashikari M. Matsuoka M. Plant Cell. 2002; 14: 57-70Crossref PubMed Scopus (401) Google Scholar), but the target sequence for the GRAS protein has not been reported. Here, we describe the isolation and characterization of a novel GRAS gene from lily microsporocytes and show that the GRAS gene product is able to activate the transcription of meiosis-associated gene by transient expression assay. Plant Materials—Flower buds of L. longiflorum cv. Hinomoto were categorized according to their length, which was calculated from the base of the pedicel to the tip of the sepals (ranging from 10 to 35 mm). Estimation of the stages of the microsporogenesis was based on the correlation between bud length and the determination of meiotic stage by cytological examination according to published methods (2Erickson R.O. Am. J. Bot. 1948; 35: 729-739Crossref Google Scholar). Anthers were isolated from the dissected buds. Leaves were collected from fresh lilies. All materials were soaked in liquid nitrogen and stored at -80 °C. DNA Sequencing—DNA sequencing was performed using a Perkin-Elmer dye primer cycle system according to the manufacturer's instructions with a ABI 373 Stretch sequencer (Applied Biosystems Inc.). Homology searches were performed in the GenBankTM data base using the BLAST program (19Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (73420) Google Scholar). RNA Isolation and Gel Blot Analysis—RNA was prepared from samples kept at -80 °C using the aurintricarboxylic acid method as described previously (20Mousavi A. Hiratsuka R. Takase H. Hiratsuka K. Hotta Y. Plant Cell Physiol. 1999; 40: 406-416Crossref PubMed Scopus (18) Google Scholar). Each lane was loaded with 10 μg of total RNA, which was then fractionated on 1% agarose-formaldehyde gels and blotted onto Hybond N+ membrane according to the manufacture's protocol (Amersham Biosciences). Hybridization of 32P-labeled DNA probes to RNA blots was performed in 50% formamide, 5% SDS, and 5× SSC. The filters were prehybridized for 2 h and hybridized overnight at 42 °C. The RNA blots were washed for 20 min in 2× SSC, 1% SDS at 42, 50, and 60 °C, briefly air-dried, and then autoradiographed with X-AR of cDNA RNA was from buds at the stage of lily PMCs by using an Plant and with (Amersham Biosciences). was performed with the system for of cDNA using the and and of the GFP-LlSCL in green fluorescent protein the from was by the of DNA for the regions of LlSCL were using the with and is and and and and These were with the appropriate and into All GFP-LlSCL fusion and fusion proteins with at the and with LlSCL at the cells were prepared and with with the were kept at for and the localization was using a fluorescent with a system in Plant transcriptional activation through the GAL4 target we used the gene of the GAL4 target sequence fused to the of the cauliflower mosaic the of the the LlSCL were fused to the GAL4 DNA-binding domain and into the by the and is and and and and and and and and and The yeast GAL4 activation domain was from with the and These were with appropriate and into the a GAL4 DNA-binding the of LlSCL protein in plant the was with and and then into by the A a was as described previously H. M. Takase H. Hiratsuka K. Plant Biotechnol. 2001; 18: Scopus Google Scholar). M. Y. and K. The the lily and PMCs were at for and h in the was as described previously N. M. T. Hiratsuka K. Plant Biotechnol. 2001; 18: Scopus Google Scholar). and a yeast expression the LlSCL and yeast activation domain were into the used in experiments were and of yeast was carried out by the The used was The liquid was performed with a according to the manufacturer's instructions Isolation of a GRAS cDNA from have and sequenced cDNA from a cDNA library of lily PMCs by the method as described previously (7Morohashi K. Takase H. Hotta Y. Hiratsuka K. Plant Biotechnol. 2000; 17: 131-135Crossref Scopus (5) Google Scholar). One of cDNA clones, designated M1125, is related in amino acid sequence to the SCR gene, which encodes a putative transcription factor that an asymmetric cell division in the Arabidopsis root (8Di Laurenzio L. Wysocka-Diller J. Malamy J.E. Pysh L. Helariutta Y. Freshour G. Hahn M.G. Feldmann K.A. Benfey P.N. Cell. 1996; 86: 423-433Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). By using the we isolated a cDNA of the from the total RNA at the stage of lily The cDNA an with a for amino of the acid sequence with the previously described proteins showed a similarity to the and SCR proteins to the GRAS protein family. of the protein, designated LlSCL (Lilium longiflorum with other GRAS gene products revealed the of highly conserved and within the carboxyl terminus of the protein referred to as and are identified in the carboxyl terminus and In the of the LlSCL protein, the which has been shown to the of transcriptional to nuclear E. S. M.G. 1997; PubMed Scopus Google Scholar, C. Gene. 2000; 245: PubMed Scopus Google Scholar), was Although no nuclear localization signal was within the LlSCL amino acid two of the highly basic referred to as and were identified and The amino terminus of the LlSCL protein not show homology to proteins of LlSCL with GRAS gene family. have been to the and and amino are in and The domain is indicated by a The shows and that of the and the are indicated by and LlSCL at the the expression of the LlSCL gene, we RNA 2 shows the results by a of total RNA prepared from collected at various stages of anther development with the of the LlSCL A LlSCL was to be regulated during the of The of LlSCL was in sporogenous cells the meiosis of microsporogenesis, the was in a and then to an in mature pollen In LlSCL gene expression were not of LlSCL and related proteins have been suggested to be involved in the transcriptional regulation (8Di Laurenzio L. Wysocka-Diller J. Malamy J.E. Pysh L. Helariutta Y. Freshour G. Hahn M.G. Feldmann K.A. Benfey P.N. Cell. 1996; 86: 423-433Abstract Full Text Full Text PDF PubMed Scopus (850) Google Scholar). they are to have nuclear In a report indicated that fusion proteins are in the of the cell (11Silverstone A.L. Ciampaglio C.N. Sun T.-P. Plant Cell. 1998; 10: 155-169Crossref PubMed Scopus (612) Google Scholar). the localization of LlSCL protein, chimeric genes the LlSCL protein fused to the were into cells using a system and expressed of the promoter green from fusion proteins were by fluorescent The fluorescent from the were in both the and the of cells the from cells with and were preferentially in the and The fusion proteins in Although of the were in both the and the in the were in the the from the were in the the from the were in the the expressed a fusion protein the These results that both and are important for the nuclear localization of LlSCL It is to the of basic regions in the of protein; however, we not out a analysis using protein because of to fluorescent from samples with The LlSCL a the function of the LlSCL protein as a transcription factor, we performed transcriptional activation experiments by transient in cells. The gene was the gene by a promoter of the GAL4 target sequence fused to the promoter The genes were to of various of the LlSCL protein with the DNA-binding domain of yeast The activation domain of yeast GAL4 protein was used as a The used and the results are shown in The LlSCL protein fused to was able to the activation the UAS promoter higher the activation of However, truncated LlSCL and fused to not activate the UAS the other the of the LlSCL protein fused to the UAS The transcriptional activation of fusion protein the were to the of the These results suggest that the amino terminus of the LlSCL protein the of strong transcriptional The amino terminus of LlSCL protein has two The domain for transcriptional activation to the sequence that is in E. M. PubMed Scopus Google Scholar, Dev. 1995; PubMed Scopus Google Scholar). we dissected the of LlSCL protein to the of transcriptional activation with to shown in which the domain showed strong transcriptional activation to that of The domain fused to to higher activity the other and showed no transcriptional both the the activity was by the expression of the domain and the These results suggest that the the domain and the domain but not the domain of LlSCL protein is for the transcriptional The of LlSCL as a in the LlSCL protein transcription through a plant-specific we the activity of transcriptional activation of LlSCL protein in yeast cells. various the GAL4 DNA-binding domain of regions of LlSCL protein in These were into yeast cells the gene the of the The results of the indicated that the of transcription activation of LlSCL protein in yeast cells were the as in plant cells. The the domain and the domain of the amino terminus of LlSCL protein transcriptional activation in the yeast as well as in the plant cell. the transcriptional activation of the fusion proteins the LlSCL domain were higher that of fusion in yeast cells. These results suggest that plant-specific factors are not required for the strong activity of transcriptional activation of LlSCL LlSCL a of the in study the LlSCL gene functions as a transcriptional at microsporogenesis, we the effect of the LlSCL protein expression on the transcriptional activity of the meiosis-associated promoter that gene a meiosis-associated gene, which encodes a molecular protein specifically induced at the meiotic in lily the transcriptional activation of the meiosis-associated the was of the promoter H. and K. in The LlSCL cDNA by the promoter was used as the The activity of the promoter was higher the meiosis-associated promoter in cells and lily the higher activity of the meiosis-associated promoter was in lily PMCs the LlSCL protein was with the activity of promoter was in plant cells the activity of the meiosis-associated promoter was by with LlSCL in cells and lily In the activity of the meiosis-associated promoter was enhanced by LlSCL the activity of promoter was These results suggest that the LlSCL protein plays a role in the transcriptional activation of the meiosis-associated promoter during meiosis in with A a GRAS at we isolated and a novel GRAS gene, LlSCL longiflorum Scarecrow-like), expressed specifically in premeiotic phase within the of L. longiflorum. the SCR has been predicted to be a transcriptional we that the LlSCL gene encodes a novel transcriptional regulator involved in The molecular experiments indicated that both the and basic regions and of the LlSCL protein are important for the nuclear localization. Although the within plant transcription factors in and is that the of the LlSCL protein the two regions by of has not been reported in previously described transcription factors GRAS gene products L. J. 1999; PubMed Scopus Google Scholar). The amino acid sequence of the domain of LlSCL protein homology with the basic in the SCR protein, but domain to of the LlSCL protein was not in the SCR In addition, no homology was between the putative of the protein and of LlSCL protein (11Silverstone A.L. Ciampaglio C.N. Sun T.-P. Plant Cell. 1998; 10: 155-169Crossref PubMed Scopus (612) Google Scholar). the other the PAT1 protein has been shown to no putative NLS, and the protein is the (13Bolle C. Konez C. Chua N.-H. Genes Dev. 2000; 14: 1269-1278PubMed Google Scholar). These data suggest the of various functions and mechanisms localization of GRAS gene of LlSCL a the GRAS gene products highly conserved within the carboxyl the amino termini were (16Pysh L.D. Wysocka-Diller J.W. Camilleri C. Bouchez D. Benfey P.N. Plant J. 1999; 18: 111-119Crossref PubMed Scopus (527) Google Scholar). The of the amino termini of GRAS gene products would be related to their In the which is in the amino termini of the and SLR1 is required for the specific functions of proteins in the gibberellin (11Silverstone A.L. Ciampaglio C.N. Sun T.-P. Plant Cell. 1998; 10: 155-169Crossref PubMed Scopus (612) Google Scholar, 18Itoh H. Ueguchi-Tanaka M. Sato Y. Ashikari M. Matsuoka M. Plant Cell. 2002; 14: 57-70Crossref PubMed Scopus (401) Google Scholar, C. Plant Cell. 2002; 14: PubMed Scopus Google Scholar). the the SCR protein, which has a function from other GRAS not in the amino The LlSCL protein not and shows no similarity to the amino terminus of SCR is that the LlSCL gene would have specific functions from the gibberellin and from asymmetric cell division in the for the the molecular functions of the amino terminus of GRAS gene products have not been reported. In however, we have demonstrated that the amino terminus of LlSCL protein is able to direct transcriptional activation to the of the activation domain of yeast GAL4 It is well that between the are associated with the transcriptional activation Dev. 1995; PubMed Scopus Google Scholar). is with the that the amino terminus of LlSCL is highly with a of The amino terminus of LlSCL protein be into and and clearly that both the and but not the show strong The transcriptional activation of both was not only in plant cells but in yeast cells. that the mechanism of transcriptional activation by the amino terminus of LlSCL protein is et al. (11Silverstone A.L. Ciampaglio C.N. Sun T.-P. Plant Cell. 1998; 10: 155-169Crossref PubMed Scopus (612) Google Scholar) a for gibberellin signal transduction through at of the LlSCL protein within the and be that the be by factors in cells and yeast the of transcriptional with the activity of transcriptional activation of the amino terminus of LlSCL protein, the LlSCL protein activity The carboxyl terminus of the LlSCL two a and an both of which is that the LlSCL protein with factors involved in the regulation of transcription protein that the of transcriptional Richards et al. D.E. J. Harberd N.P. 2000; PubMed Scopus Google Scholar) that GRAS gene products were related to the family of proteins based on structural similarities between GRAS and family are by the J.E. Science. 1997; PubMed Scopus Google Scholar), the LlSCL protein be by an protein for a the and are in cells 1997; PubMed Scopus Google Scholar), be to the of LlSCL proteins with amino acid within the LlSCL a in during the experiments of transient expression of the LlSCL protein in plant we that the LlSCL protein activate the expression of the meiosis-associated gene in lily PMCs is the report that the GRAS gene product is involved in regulation of a specific gene with the of we report for the that a GRAS gene is involved in the specific gene expression during The results in study indicated that the LlSCL protein a specific for specific activation in because the LlSCL protein not the of promoter in and the LlSCL protein functions as a for activating a gene expression with the specific elucidate the mechanisms involved in gene is to the specific that activate gene expression in with the LlSCL Further studies be to the functions and of LlSCL protein in the microsporogenesis and anther development in However, because of to a molecular studies using a lily system are The GRAS gene, which shows a similarity to LlSCL gene, would have a function in the from various Recently, we identified a gene the LlSCL from Arabidopsis and H. and K. The of LlSCL from species would study of the function and of the LlSCL