Botany Publication and Papers Botany 6-1997 Cloning, mapping, and analyses of expression of the Em-like gene family in soybean [Glycine max (L). Merr.] E. S. Calvo Iowa State University E. S. Wurtele Iowa State University, [email protected] R. C. Shoemaker United States Department of Agriculture Follow this and additional works at: http://lib.dr.iastate.edu/bot_pubs Part of the Agronomy and Crop Sciences Commons, Botany Commons, Cell and Developmental Biology Commons, and the Plant Breeding and Genetics Commons Recommended Citation Calvo, E. S.; Wurtele, E. S.; and Shoemaker, R. C., "Cloning, mapping, and analyses of expression of the Em-like gene family in soybean [Glycine max (L). Merr.]" (1997). Botany Publication and Papers. 67. http://lib.dr.iastate.edu/bot_pubs/67 This Article is brought to you for free and open access by the Botany at Iowa State University Digital Repository. It has been accepted for inclusion in Botany Publication and Papers by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Theor Appl Genet (1997) 94 : 957Ð967 ( Springer-Verlag 1997 E. S. Calvo á E. S. Wurtle á R. C. Shoemaker Cloning, mapping, and analyses of expression of the Em -like gene family in soybean [Glycine max (L). Merr.] Received: 25 September 1996 / Accepted: 27 September 1996 Abstract The entire Em-like Group-1 late embryo- Key words Em-like gene family á Soybean á genesis abundant (Lea) gene family from soybean was Cloning á Characterization á Mapping cloned and characterized. The five Group-1 Lea genes (Sle1-5) were divided into two classes based on se- quence identity. Sle1-4 were genetically mapped to four di¤erent linkage groups. Nucleotide sequencing in- Introduction dicated that Sle1, Sle2, Sle3, and Sle5 encode polypep- tides di¤ering primarily by the presence of a repeated A considerable amount of information has accumu- 20-amino acid motif. Sle1 and Sle5 were shown by lated on the molecular biology of the events involved in Northern analysis to be expressed in developing em- the developmental regulation of expression of several bryos weeks earlier than Sle2 and Sle3. Sle4 was shown seed-protein genes, particularly those encoding storage to be a pseudogene. Maximal levels of mRNA for all proteins (Goldberg et al. 1989; Goldberg et al. 1994). functional Sle genes accumulated in maturation-phase Among these are included the Lea (late embryogenic seeds, before significant desiccation had occurred, and abundant) genes (Dure 1993). LEA polypeptides can be declined rapidly upon seed imbibition. Desiccation did grouped into at least three structurally distinct groups not induce Sle expression in seeds or vegetative tissue. based on amino acid sequence homology and occur- Sle expression was confined to embryo tissues and Sle rence of amino acid motifs (Dure et al. 1989). The mRNA accumulated at similar levels in both the em- isolation of homologous genes or cDNAs has been bryo axis and in the cotyledons. reported from several species (Almoguera and Jordano 1992; Espelund at al. 1992; Litts et al. 1992; Raynal et al. 1989; Ulrich et al. 1990; Williams and Tsang 1991; Wurtele et al. 1993), but none of these reports men- tioned genes or cDNAs from legume species. Joint contribution of North Central Region, USDA-ARS and Jour- nal Paper No.17317 of the Iowa Agricultural and Home Economics It has been hypothesized that the LEA proteins are Experiment Station, Ames, Iowa, 50011. Project 3236. Names are involved in protecting plant cells from the harms of necessary to report factually on available data; however the USDA desiccation (Dure et al. 1989). Support for this hypothe- neither guarantees nor warrants the standard of the product and the sis comes mostly from circumstantial evidence, such as use of the name by the USDA implies no approval of the product to abundance in the cell, the time of LEA protein appear- the exclusion of others that may also be suitable. ance during development, and their hydrophilic nature Communicated by A. L. Kahler (Dure 1993). Experimental evidence is limited to bi- ophysical measurements of hydrodynamic properties of E. S. Calvo ( ) the wheat Em protein (McCubbin et al. 1985). Com- Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011, USA puter modeling of Group-3 proteins has predicted a tertiary structure compatible with the hypothesis that E. S. Wurtele Department of Botany, Iowa State University, Ames, such proteins would be involved in ion sequestration in Iowa 50011, USA the desiccated cell (Dure 1993). Whether or not such a structure occurs in vivo remains to be determined. R. C. Shoemaker Department of Zoology and Genetics, and Department The recent finding of Lea genes in cyanobacteria, an of Agronomy and USDA-RRS-FCR, Iowa State University, Ames, organism more easily studied genetically than higher Iowa 50011, USA plants, will certainly contribute to our understanding of 958 the functions of LEA proteins in late embryo develop- Water-deficit treatments ment (Curry and Walker-Simmons 1993). Much of our information on the regulation of Lea Seeds containing embryos at 30 DAF were left in excised pods and gene expression has been derived from experiments allowed to dry for 48 h (Rosenberg and Rinne 1988). Treated em- bryos (1Ð2 g fresh weight) were harvested, frozen in liquid nitro- using monocot species (Marcotte et al. 1989; McCarty gen, and stored at !80¡C. For treatment of seedlings, germination and Carson 1991; McCarty et al. 1991). However, this papers containing 4-day-old seedlings were removed from the information can not necessarily be extrapolated to water-containing tray, unfolded, laid over dry trays, and kept dicots (Galau et al. 1991). Unlike monocots, where in a cabinet for 26 h to ensure a slow dehydration. Tissues of storage-protein gene expression is frequently concen- the seedlings were visibly dehydrated as judged by the turgor of the hypocotyl. Seedlings in which the root tip was completely dry trated in the endosperm, the embryo cotyledons and to were discarded. Roots, hypocotyls, and cotyledons of 10Ð15 seed- some extent the embryo axes are the primary tissue for lings were harvested and immediately frozen in liquid nitrogen and storage-protein gene expression in seeds of the majority stored at !80¡C. of dicot species (Lopes and Larkins 1993). This is parti- For the water-deficit treatment of plantlets, shoots of 5-week-old plants were harvested, weighed, and left on the laboratory bench for cularly true for legumes such as soybean where the about 6.5 h, or until shoots had lost 20% of their initial weight endosperm is very short lived (Chamberlin et al. 1994) (Plant et al. 1991). Leaves from 12 plants were harvested, pooled, and where storage proteins account for 36% or more of frozen in liquid nitrogen, and stored at !80¡C. the seed dry weight (Wilson 1987). As a consequence, in soybean both the embryo-specific maturation and late embryogenesis programs may take place exclusively in Isolation of cDNA and genomic clones the embryo. Therefore, it is conceivable that embryos of legume species may utilize a somewhat di¤erent mecha- A lgt11 soybean (cv ‘Enrei’) cDNA library, constructed from mid- nism from monocots to achieve the temporally di¤erent maturation developing seeds, was kindly provided by Drs. D. Shibata and R. Whittier (Mitsui Plant Biotechnology Research expression of the maturation and late embryogenesis Institute, Tsukuba, Japan). Bacteriophage manipulation and programs. screening techniques were as described by Sambrook et al. (1989). Previous studies have shown that members of multi- Nitro-cellulose (Schleicher & Schuell) filters were pre-hybridized gene families in soybean pair into more closely related and hybridized in 6]SSC (1]SSC is 0.18 M NaCl and 1.9 mM groupings (Grandbastien et al. 1986; Lee and Verma sodium citrate, pH 7.0), 1% (w/v) SDS, 25 mM NaHPO4, 1984; Nielsen et al. 1989). The analysis of members pH 6.5, 3]Denhardt’s, and 0.1 mg/ml herring sperm DNA, at 65¡C. Final washes were done in 0.5]SSC, 0.1% SDS, at 56¡C, of a gene family can provide much information on for 1 h. The probe used for screening the library was the carrot evolutionary processes and genome organization, EMB-1 cDNA (Ulrich et al. 1990) labeled with a-[32P]-dCTP and gene expression. The primary objective of the (Feinberg and Volgelstein 1983). The cDNAs hybridizing to the research presented here was to clone, sequence, and EMB-1 probe were subcloned into the EcoRI site of pBluescript KS` (Stratagene). analyze the expression of the complete Em-like Genomic clones for Sle1 and Sle4 were isolated from a lEMBL3 gene family from soybean. This would provide the genomic library made from cv ‘Williams’ (Clontech), and those framework for studing the gene-family evolution and for Sle2 and Sle3 were isolated from a lEMBL3 library made from regulation of Lea gene expression during soybean cv ‘Resnik’ (Clontech). The Sle5 clone was isolated as a 2.5-kb seed development. This report identifies and character- EcoRI fragment from a lgt10 genomic library made after gel-frac- tionating (2.3Ð3.5 k b) EcoRI-digested DNA (cv ‘Williams’) in izes all five members of the Group-1 Lea gene family low-melting agarose (FMC). DNA was recovered from the gel using from soybean and genetically maps the location of four a QIAGEN tip-5 column essentially as described by the manufac- of these genes. turers. Library construction followed procedures described in Sambrook et al. (1989). The three genomic libraries were screened with the soybean cDNA. Screening conditions were as described for the cDNA library except that the final wash was done at 2]SSC, 0.1% SDS, at 50¡C.