Copyright 0 1996 by the Genetics Society of America Dosage Effects on Gene Expression in a Maize Ploidy Series Mei Guo, Doug Davis and James A. Birchler Division of Biological Sciences, University of Missouri, Columbia, Missouri 6521 1 Manuscript received August 24, 1995 Accepted for publication December 22, 1995 ABSTRACT Previousstudies on gene expression in aneuploidsrevealed numerous trancacting dosage effects. Segmental aneuploidy of each varied chromosomal region exhibited predominantly inverse effects on several target genes. Here, dosage regulation was examined in a maize (&a mays L.) ploidy series where the complete genomic complementis vaned. TotalRNA from leaf tissue of monoploid, diploid, triploid, and tetraploid plants (lx, 2X, 3X, and 4X, respectively) was analyzed for the expression of 18 genes. For most tested genes, the transcript level per cellis directly proportional to structural gene dosage; that is, on a per genome basis, thereis approximately equal expression among the four ploidies. Exceptional cases show a negative correlation of expression with ploidy aor positive correlation greater than expected from the structural gene dosage. These studies suggest that, in asgeneral, structural gene dosage increases in multiples of the monoploid complement, the absolute levelof gene expression per cell increases. In contrast, addition or subtractionof only a single chromosome arm tends to alter gene expression patterns extensively. The combined results of the euploid and aneuploid studies suggest that aneuploid effects result from an altered stoichiometry of the factors contributing to the mechanismsof gene expression. HE gene dosage changes affecting structural gene In addition, when the dosage of a chromosomal seg- T expression have been studied in several species ment is varied, trans-acting effects, either positive (di- including human, mouse, Drosophila and plants rect) or negative (inverse), on genes elsewhere in the (BIRCHLER1979, 1981; BIRCHLERand NEWTON 1981; genome have been observed. We previously studied WHATLEYet al. 1984; EPSTEIN1986; REICHERT 1986; DEV- gene dosage effects of maizeaneuploids using B-A trans LIN et al. 1988; BIRCHLERet al. 1990; GUOand BIRCHLER locations to add or subtract single chromosome arms 1994). Changes in dosage include an aneuploid series relative to normal (GUO and BIRCHLER1994). Expres in which a chromosome or chromosomal segment is sion patterns of six genes were examined in a dosage added or subtracted relative to the genomic comple- series of 14 chromosomal segments. Transcript levels of ment or a ploidy series where a complete genomic in- each tested gene were affected by multiple chromo- crement is increased or decreased. When the dosage of somal regions. Each varied chromosomal region showed a structural gene is varied, typically two types of dosage trans-effects on several genes. Such modulations are pre- regulation on its expression are observed. One is a gene dominantly inverse (negative) effects on gene expres- dosage effect where there are increases or decreases in sion. When the dosage of a transacting inverse regulator expression in proportion to the gene copy number. of a target gene is varied simultaneously with the stmc- Gene dosage effects are often observed (e.g., GRELL tural gene, the dosage effect of the latter is cancelled 1962; EPSTEIN1986). Thisis particularly the case when by the former, resulting in the compensation described theaneuploid segments surroundingthe structural above (BIRCHLER1981; GUOand BIRCHLER1994). geneare small (BIRCHLER1981). Theother typeof Because of thepredominant lethality of ploidy dosage regulation is called dosage compensationin changes in animal systems, most ofthe studies on dosage which there is an equal expression regardless of the regulation ofploidy series have been conducted on copy number of the respective gene in aneuploids in- plants, although a few studies on yeast, Drosophila and volving significant cytological length. Studies of such animal cell lines have been reported (CIFERRIet al. 1969; dosage series from various organisms, especially Dro- PRIESTand PRIEST1969; LUCCHESIand RAWS 1973). sophila and plants, reveal that gene expression is often Among isozymesand proteins examined in various plant dosage compensated (BIRCHLER1979; BIRCHLERand species, some are positively or negativelyaffected by NEWTON 1981).This phenomenon is found with X ploidy changes, while most ofthem exhibit an increased chromosome linked genes,as well asgenes encoded on level as the ploidy level rises,that is, a gene dosage effect the autosomes of Drosophila (MULLER1932; DEVLINet (DEMAGGIOand LAMBRUKOS 1974;LEVIN et al. 1979; al. 1982; BIRCHLERet al. 1990). TIMKOet al. 1980; BIRCHLERand NEWTON1981). Mor- phologically documented effects withincreased ploidy in Curresponding author: James A. Birchler, Division of Biological Sci- ences, 117 Tucker Hall, University of Missouri, Columbia, MO 6521 1. plants include increased cell size and organ size while E-mail: [email protected] the vigor decreases beyond the tetraploid level (RHOADES Genetics 142: 1349-1355 (April, 1996) 1350 M.Davis Guo, D. and J. A. Birchler and MCCLINTOCK1935; BLAKESLEE1941; RHOADES and ploids and hexaploids fail to germinate orsurvive beyond the DEMPSEY1966). No previous studies have examined spe- early seedling stage. Plants of the ploidy series were transferred from the green- cific RNA levels in a ploidy series. house to the field after cytological confirmation by root tip In the present study, we investigated gene dosage chromosome counts. At the six-leaf stage the younger four effects further by varying the ploidy, where genomic leaves of each plant were harvested. The tissue was frozen in balance is maintained as compared with aneuploids. liquid nitrogen and stored at -80” until use. We sought to test whether the aneuploid effects were RNA isolation: Leaf tissue from single plants of each ploidy was used for RNA preparations. Total RNA was extracted us- cumulative, in principle, or whether a “balance” was ing amodified method of CONE et al. (1986). Tissue was operating as suggested by classical phenotypic observa- ground to a fine powder in liquid nitrogen with a mortar and tions. The transcript levels of 18 genes expressed in leaf pestle. Cells were lysed in 2.5-3.0 volumes of buffer con- tissue were analyzed inmonoploid, diploid, triploid and taining 0.1 M NaCl, 50 mM Tris-HC1 (pH 7.4),50 mM disodium tetraploid maize plants. Most genes exhibited a gene ethylenediaminetetraacetate (EDTA), 2% sodium dodecyl sul- fate (SDS) and proteinase K (200 ,ug/ml). The supernatant dosage effect relativeto the ploidy level,although inter- was extracted twice with an equal volume of pheno1:chloro- esting exceptions occurred in which a strong positive form:isoamyl alcohol (100:99:1) and twicewith chloro- correlation or a negative correlation occurred between form:isoamyl alcohol (99: 1). Total RNA was precipitated in a gene expression and ploidy. final concentration of 2 M LiCl solution and reprecipitated with 2.5 volumes of absolute ethanol and one-tenth volume of 3.0 M sodium acetate (pH 5.0). MATERIALSAND METHODS Probe sources and Northern analysis: Except for the three well-characterized genes, Alcoholdehydrogenase 1 (Adhl),Alcohol Genetic stocks and production of a ploidy series: The dip- dehydrogenase 2 (AdhP) and Sucrosesynthase (Susl),those exam- loid (Oh 40B) and tetraploid (Alexander synthetic B) stocks ined were mapped isolates derived from a maize leaf cDNA were obtained from the Maize Genetics Cooperation. The library and were provided by the University of Missouri, Co- 1X -4X ploidy series was generated as follows: The monoploid lumbia (UMC) Maize RFLP laboratory. All clones for probe was produced by using the indeterminate gametophyte (ig)muta- synthesis were in or subcloned into theBluescript vector (Stra- tion, which causes the production of androgenetic haploids tagene) , which contains T3 and T7promoters. Antisense RNA probes were prepared by in vitro transcription of the respective when used as a female parent (KERMICLE 1969). The ig stock is homozygous for R-nj, which conditions anthocyanin devel- genes in such constructs using either the T3 or T7 promoter. opment in the anther, endosperm andembryo. This line was Five micrograms of total RNA from each sample was loaded crossed by the Oh 40B/elongate (el)diploid male (see below). in each lane and subjected to electrophoresis in 1.5% formal- The androgenetic haploid was selected based on the lack dehyde-agarose gels. RNAwas then capillary transferred to of pigmentation in the embryo. Triploids were produced by Biotrans nylon membrane (ICN) and cross linked by UV irra- crossing the tetraploid with the diploid. Each ploidy was con- diation. Blots were prehybridized for 6 hr and thenhybridized firmed by cytological examination of the chromosome num- with 3’‘P labeled antisense RNA of the respective genes. The ber in root tips. hybridization conditions were 60” in 5X SSC (1X SSC consists The homozygous elongate female produces normal haploid of 0.15 M NaCl, 0.015 M sodium citrate), 50% formamide, 1OX as well as diploid gametes (RHOADES and DEMPSEY1966). The Denhardt’s, 0.5% SDS, 10% dextran sulfate and 0.2 mg/ml el stock was crossed as a female parent with the Oh40B dip- salmon sperm DNA, for -16 hr. Theblots were washed at 75” loid. Normal (diploid embryos) as wellas defective kernels with 0.2X SSC and 0.05% SDS (BIRCHLERand HIEBERT1989). (triploid embryos) are produced. The normal kernels