Genetics: Early Online, published on July 27, 2016 as 10.1534/genetics.116.191833 MATERNAL GAMETOPHYTE EFFECTS ON SEED DEVELOPMENT IN MAIZE Antony M. Chettoor†*, Allison R. Phillips†*1, Clayton T. Coker*, Brian Dilkes‡, and Matthew M. S. Evans* *Department of Plant Biology, Carnegie Institution for Science, Stanford, CA 94305, USA ‡Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA 1 Present address: Biology Department, Wisconsin Lutheran College, Milwaukee, WI 53226, USA †These authors contributed equally to this work. 1 Copyright 2016. running title: maternal effects in maize keywords: maize, embryo sac, maternal effect, endosperm transfer layer, gametophyte Corresponding author: Matthew Evans, Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, CA 94305; Phone: (650) 739-4283; Fax: (650) 325-6857; Email: [email protected] 2 ABSTRACT Flowering plants, like placental mammals, have an extensive maternal contribution towards progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization which produces sister progeny, the endosperm and the embryo, of the seed. Because of this there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays ssp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo although some embryo specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed. 3 INTRODUCTION The process of double fertilization is unique to flowering plants and results in the formation of the seed. The two sperm cells of the pollen grain fertilize the egg and central cell of the female gametophyte, or embryo sac, to form the diploid (1 maternal: 1 paternal) embryo and typically triploid (2 maternal: 1 paternal) endosperm, respectively (SHERIDAN and CLARK 1994; WALBOT and EVANS 2003). The endosperm is thus a genetic sister of the embryo and is functionally equivalent to the mammalian placenta, acting as a nutritive tissue that supports the growth of the developing embryo and seedling. The maize endosperm consists of several morphologically and transcriptionally distinct domains: the aleurone (AL), the basal endosperm transfer layer (BETL), the starchy endosperm (SE), the conducting zone (CZ), the basal intermediate zone (BIZ), and the embryo-surrounding region (ESR) (LEROUX et al. 2014; LI et al. 2014; OLSEN 2004; OLSEN et al. 1999). Haploid gene expression and patterning of the female gametophyte prior to fertilization can significantly affect the development of both the endosperm and embryo (DREWS et al. 1998; MARTON et al. 2005; VERNOUD et al. 2005; WALBOT and EVANS 2003). The maize embryo sac is produced from a single megaspore by three rounds of free nuclear divisions generating an 8- nucleate syncytium which then cellularizes to produce seven cells of four types (EVANS and GROSSNIKLAUS 2009): the egg cell, two synergids, the central cell, and three antipodal cells. Division of the antipodal cells, associated with auxin signaling, produces a cluster of 20-100 antipodal cells in maize (CHETTOOR and EVANS 2015). The function of the antipodal cells is undetermined but they are hypothesized to act as a transfer tissue based on the presence of cell wall invaginations on the surfaces facing the maternal nucellus (DIBOLL 1968). Alternatively, they could act as a signaling center providing positional information for the embryo sac, or even for the endosperm since they persist in the maize seed after fertilization (RANDOLPH 1936; WEATHERWAX 1926). 4 Two types of maternal effect, seed development mutants can be distinguished based on their mode of inheritance: those in genes required in the maternal sporophyte (LI and BERGER 2012; LI and LI 2015) and those in genes required in the maternal female gametophyte (LUO et al. 2014). They can be distinguished from each other by the mode of transmission (EVANS and KERMICLE 2001; GROSSNIKLAUS and SCHNEITZ 1998). Recessive maternal sporophyte effect mutants will only have consequences if parent plants are homozygous. Both maternal gametophyte effect mutants and dominant maternal sporophyte effect mutants produce abnormal seeds when heterozygotes are crossed as females, but in the case of gametophyte mutants the abnormal seeds inherit the mutant allele because the embryo sac must carry the mutation to cause an effect, while the allele present in the embryo sac (and hence the seed) is irrelevant in the case of maternal sporophyte effects. Consequently, in the case of dominant maternal sporophyte effects, wild-type and mutant alleles are equally represented in both abnormal and normal seeds. Gametophytic maternal effect mutants have been identified in both Arabidopsis and maize (EVANS and KERMICLE 2001; GAVAZZI et al. 1997; GRINI et al. 2002; GUTIERREZ- MARCOS et al. 2006b; KOHLER and GROSSNIKLAUS 2005; OLSEN 2004; PAGNUSSAT et al. 2005; PHILLIPS and EVANS 2011; PIEN and GROSSNIKLAUS 2007). Although not affecting post- fertilization seed development when transmitted through the pollen, many gametophytic maternal effect mutations in Arabidopsis (BOAVIDA et al. 2009; PAGNUSSAT et al. 2005) and maize (EVANS and KERMICLE 2001; GUTIERREZ-MARCOS et al. 2006b; PHILLIPS and EVANS 2011) have reduced male transmission, indicating a separate role for the gene in pollen development/function. Studies of these mutants have revealed several causes for maternal effects, as identified through genetic and cellular analysis. Maternal gametophyte effects can be caused by defects in: functional gene dosage in the endosperm (SINGLETARY et al. 1997); embryo sac morphology (LIN 1978); cytoplasmic storage of gene products (SPRINGER et al. 2000); and imprinting (KINOSHITA et al. 1999; VIELLE-CALZADA et al. 1999). Sporophytic maternal effects can occur through disruption of maternal transfer tissues or integuments (FELKER et al. 1985; GARCIA et al. 2005), non-reduction of gametes (which lead to endosperm parental ploidy imbalance) (BARRELL and GROSSNIKLAUS 2005; SINGH et al. 2011), or microRNA production (GOLDEN et al. 2002). 5 Although the two types of maternal effects have distinct modes of inheritance and time of action, there is evidence of interaction between the imprinting pathway (typically a gametophyte effect) and maternal sporophyte effects (DILKES et al. 2008; FITZGERALD et al. 2008). Non-equivalence of the maternal and paternal genomes in endosperm development was identified through the analysis of interploidy crosses (e.g. tetraploid by diploid) in multiple species of plants, and these data contributed to the formation of the parental conflict theory (HAIG and WESTOBY 1989). According to this theory, maternal and paternal alleles in the endosperm have different activities, leading to restriction or promotion of the growth of the endosperm, respectively. The endosperm phenotypes of seeds with maternal or paternal genome excess are in agreement with this theory (CHARLTON et al. 1995; HAIG and WESTOBY 1991; SCOTT et al. 1998). In maize, the BETL is particularly sensitive to maternal or paternal genome excess (CHARLTON et al. 1995). Non-equivalent expression of the parental alleles of many genes is present in the embryo as well, primarily before the mid-globular stage (AUTRAN et al. 2011; BAROUX et al. 2001; BAROUX and GROSSNIKLAUS 2015; GRIMANELLI et al. 2005; VIELLE- CALZADA et al. 2000). Analysis of the early phenotypes of embryo lethal mutants corroborated these studies and demonstrated that early embryogenesis is largely under maternal control (DEL TORO-DE LEON et al. 2014). RNA-Seq has enabled the identification of hundreds of genes with parent-specific and parent-biased expression in the seed of several plant species (GEHRING et al. 2011; HSIEH et al. 2011; PIGNATTA et al. 2014; WATERS et al. 2011; WOLFF et al. 2011; XIN et al. 2013; ZHANG et al. 2011). Many genes have only a subset of their naturally occurring alleles imprinted. Frequently, imprinting is stage-specific, with expression being uniparental early in endosperm development and biallelic later. Gametophytic maternal effect mutants in Arabidopsis frequently show defects during this early period of development (NGO et al. 2012; PAGNUSSAT et al. 2005). The imprinted status of these genes is regulated, at least in part, by parent-specific DNA methylation, Polycomb Group mediated repression, and small RNA pathways (FITZGERALD et al. 2009; GUTIERREZ-MARCOS et al. 2004; GUTIERREZ-MARCOS et al. 2006a; HAUN and SPRINGER 2008; HSIEH et al. 2011; JAHNKE and SCHOLTEN 2009; KOHLER et al. 2003; KOHLER 6 et al. 2005; MAKAREVICH et al. 2008; PIGNATTA et al. 2014; VU et al. 2013; WOLFF et al. 2011; ZHANG et al. 2014) and is often associated with repetitive DNA elements (GEHRING et al. 2009; PIGNATTA et al. 2014; VILLAR et al. 2009). Molecular mechanisms that mark and maintain silenced alleles include a complex interplay between DNA methylation and histone modifications (KAWASHIMA and BERGER 2014). While no functional data is available for most imprinted genes in plants, the maize maternally expressed meg1 gene has been shown to promote nutrient allocation to the seed by promoting differentiation of the BETL (COSTA et al. 2012). However, the promotion of endosperm growth by a maternally active gene is the opposite of that predicted by parental conflict theory and demonstrates that there is maternal control of essential seed developmental processes unrelated to parental conflict theory. A different explanation for the function of imprinting in the seed is to generate functional diversity of genes in seed development (BAI and SETTLES 2014; PIGNATTA et al. 2014). As these models are not mutually exclusive, selective pressure from both mechanisms (and others) could be operating during evolution to generate parent-of-origin specific expression of genes for different purposes in the seed. For example, some paternally expressed genes are important for establishing interploidy crossing barriers (KRADOLFER et al. 2013; WOLFF et al. 2015), while others are important for patterning of the embryo (BAYER et al. 2009; COSTA et al. 2014). Most maternal effect mutants described in Arabidopsis do not have any prefertilization morphological defects (GRINI et al. 2002; PAGNUSSAT et al. 2005), except for those with fertilization independent seed development (CHAUDHURY et al. 1997; GROSSNIKLAUS et al. 1998; KIYOSUE et al. 1999; OHAD et al. 1996). Some of the maternal effect mutants in maize have abnormal gametophyte morphology that may contribute to their effects on seed development and pollen transmission (GUTIERREZ-MARCOS et al. 2006b; PHILLIPS and EVANS 2011). Here we describe a set of maternal effect mutants in maize with varying effects on seed development. A majority have visible morphological defects in the embryo sac before fertilization, and an overlapping majority affect patterning of BETL gene expression in the endosperm after fertilization. In most cases, the pre-fertilization defects are sufficient to explain the defects in seed development. Consequently, only a subset of these mutations may affect 7 imprinted genes or imprinting processes. Whether or not any of these mutations have imprinting-specific effects or affect both imprinting and pre-fertilization embryo sac development will be resolved after cloning and molecular analysis of the affected genes. MATERIALS AND METHODS Plant material and growth conditions: This collection of maize maternal effect mutants (mem) was developed from a variety of mutagenesis populations as follows. The Mn-Uq mutant was isolated previously (PAN and PETERSON 1989). The sans scion1 (ssc1), heirless1 (hrl1), no legacy1 (nol1), baseless2 (bsl2), and topknot1 (tpn1) mutants were identified as rare ears with 25-50% defective kernels after pollination of females with wild-type males during routine propagation of maize genetic stocks. The ssc1 mutation arose in a W22 inbred maize (Zea mays) plant carrying a mutable allele of enhancement of r1 (enr1) and a pale-aleurone-conferring allele, R1-r::(Venezuela), of the r1 gene (STINARD et al. 2009). hrl1 arose in a W64A inbred line with active Mutator (Mu) transposons. nol1 arose in a line with active Ac/Ds transposons from a seed carrying a revertant to wild type of a vp1-m1::Ds mutant allele. bsl2 arose in an active Mu W64A/A158 hybrid line. topknot1 (tpn1) arose in an active Mu B73/W23 hybrid line. Two mutants, superbase1 (sba1) and maternally reduced endosperm1 (mrn1), were identified from an EMS mutagenesis as rare ears with a high frequency of defective kernels in an open pollinated population. One mutant, hrl2, arose as a single defective kernel event in a W22 inbred line with active Ac/Ds transposons. The other mutants arose in UniformMu maize lines, inbred W22 (MCCARTY et al. 2005), as single defective kernel events on otherwise wild-type ears. Mutants were typically propagated as heterozygotes by transmission through the female and selection for miniature or defective kernels. Mutants and wild-type controls were grown side by side for each experiment either in summer field conditions or in greenhouses under long-day conditions (16 hour light : 8 hour dark cycles). 8 Most mapping populations were generated by crossing mem/+Mo17 or mem/+B73 hybrid females by wild-type Mo17 or B73 males, respectively. For hrl1 and bsl2, the mutant phenotype was suppressed in F1 hybrids with B73 and Mo17, so the mapping populations were generated by crossing mem/+B104 females by wild-type B104 males. For hrl2, the mapping population was generated by crossing hrl2W22/+W64A females by wild-type W64A males. Molecular mapping: DNA was extracted from seedlings by minor modification of the method of (SAGHAI- MAROOF et al. 1984) or from mature seeds (MARTIN et al. 2010), and PCR reactions were performed as described (EVANS and KERMICLE 2001). Initial map position was determined from Bulk Segregant Analysis (BSA), comparing DNA from a pool of 48 normal seeds (mostly wild- type homozygotes) to DNA from a pool of 48 defective seeds (mutant heterozygotes), using either SNP-based Sequenom mapping (LIU et al. 2010) or PCR with a set of polymorphic SSR markers (MARTIN et al. 2010). When BSA showed heterozygosity in the mutant pool but near homozygosity in the wild-type pool, PCR was performed using the same SSR markers or nearby SSR markers on 48 defective and 48 normal kernel individuals to verify co-segregation with the mutant phenotype. Map position was refined using additional SSR and IN/DEL PCR based markers within the chromosomal interval identified that showed polymorphisms between the two parental lines. Transmission and viability assays: For ssc1, map position was first identified based on linkage to the visible kernel mutant yellow endosperm1 (y1). Male and female transmission of the ssc1 mutation and penetrance of the defective kernel phenotype were partially assessed using plants carrying ssc1 linked in repulsion phase to y1. The genetic distance between ssc1 and y1 was determined using the kernel phenotypes of y1 and ssc1. Transmission of y1 was observed after making reciprocal crosses between + y1/ssc1 + plants and homozygous y1 plants. Similarly, for stt3 and mrn2, map position was identified based on linkage to the r1 gene. Male and female transmission of stt3 and mrn2 and penetrance of the defective kernel phenotype were partially assessed using reciprocal crosses between heterozygous mem R1-r::standard/+ r1-r plants and homozygous r1- r plants. For all other mutants, normal kernels from reciprocal crosses between mutant 9 heterozygotes and wild-type plants were grown to maturity and progeny tested to determine what fraction had inherited the wild-type allele (i.e. were homozygous wild type) and what fraction had inherited the mutant allele (i.e. were heterozygous). For male crosses this frequency produces the male transmission rate. For the female crosses this frequency is combined with the frequency of the defective kernels to calculate the female transmission rate using the percentages of all kernels that are homozygous wild type, all that are defective, and all that are heterozygous mutant but appear normal. To calculate the percentage of embryo sacs carrying the mutation that produced a detectable kernel (whether defective or normal), it was assumed that half of the embryo sacs inherited the mutation. If fewer than half of all kernels were mutant heterozygotes, then the number of embryo sacs that would need to be added to make the number of homozygous wild type and heterozygous mutant kernels equal was assumed to consist of mutant embryo sacs that did not produce a detectable kernel. For viability assays, defective kernels, regardless of severity, were germinated on filter paper. If necessary, growing shoot tips were liberated by making an incision in the pericarp just beyond the tip of the shoot. Seeds with any root or shoot growth were transplanted to soil in small pots, and survivors were transplanted at the 2-3 leaf stage to the field and grown to maturity. Confocal microscopy and histology: Embryo sacs were analyzed from mutant heterozygotes at mature stage (with a silk length ≥20cm). Samples were processed and visualized on a Leica SP5 or Leica SP8 (Wetzlar, Germany) laser scanning confocal microscope as described previously (GUTIERREZ-MARCOS et al. 2006b; PHILLIPS and EVANS 2011). Excitation was performed at 405 nm, 488 nm, and 561 nm and emission was collected at 410-480 nm, 495-555 nm, and 565-730 nm for the merged images. Images were analyzed and processed using ImageJ (NIH) and Adobe Photoshop CS3. Figures were produced by generating a single image from a projection of all optical sections containing embryo sac nuclei. For the reporter assays, mutant heterozygotes were crossed as females by males either hemizygous or homozygous for ProBet1::b-glucuronidase (GUS) (HUEROS et al. 1999). For some mutants, additional GUS assays were performed on kernels from crosses of females heterozygous for the mutation and hemizygous for the Bet1::GUS transgene after pollination Pro 10