Maize ROP2 GTPase Provides a Competitive Advantage to the Male Gametophyte

Corresponding author: J. E. Fowler, 2082 Cordley Hall, Oregon State University, Corvallis, OR 97331-2902., fowlerj{at}science.oregonstate.edu (E-mail)

Communicating editor: J. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Rop GTPases have been implicated in the regulation of plant signal transduction and cell morphogenesis. To explore ROP2 function in maize, we isolated five Mutator transposon insertions (rop2::Mu alleles). Transmission frequency through the male gametophyte, but not the female, was lower than expected in three of the rop2::Mu mutants. These three alleles formed an allelic series on the basis of the relative transmission rate of each when crossed as trans-heterozygotes. A dramatic reduction in the level of ROP2-mRNA in pollen was associated with the three alleles causing a transmission defect, whereas a rop2::Mu allele that did not result in a defect had wild-type transcript levels, thus confirming that mutation of rop2 causes the mutant phenotype. These data strongly support a role for rop2 in male gametophyte function, perhaps surprisingly, given the expression in pollen of the nearly identical duplicate gene rop9. However, the transmission defect was apparent only when a rop2::Mu heterozygote was used as the pollen donor or when a mixture of wild-type and homozygous mutant pollen was used. Thus, mutant pollen is at a competitive disadvantage compared to wild-type pollen, although mutant pollen grains lacked an obvious cellular defect. Our data demonstrate the importance in vivo of a specific Rop, rop2, in the male gametophyte.

SELECTION can take place throughout both the sporophytic and gametophytic generations. However, during the male gametophytic phase, in particular, large haploid populations often compete to fertilize the ovules, resulting in the potential for increased selective pressure on the male gametophyte as compared to that on the sporophyte (MULCAHY and MULCAHY 1987 ). Genetic differences that affect pollen development and function (e.g., germination ability or pollen tube growth rate) contribute to variability in pollen fitness. These differences among individual gametophytes can lead to nonrandom fertilization and thus affect the population genetic structure (MULCAHY et al. 1996 ; SKOGSMYR and LANKINEN 2002 ). Furthermore, selection in the gametophytic generation can influence the sporophytic generation (OTTAVIANO et al. 1988 ), a phenomenon thought to be due to the numerous genes that are expressed in both generations (TWELL 1994 ; HONYS and TWELL 2003 ). Little is known about the molecular genetic basis of male gametophytic development, particularly regarding those genes that act in the later stages and that might encode molecules that influence pollen competitiveness (FRANKLIN-TONG 2002 ; JOHNSON and PREUSS 2002 ; LORD and RUSSELL 2002 ; SARI-GORLA et al. 2002 ).

Pollen development and function are most likely controlled by a large number of genes. Although mRNA populations in maize pollen are less complex than those in shoots, WILLING et al. 1988 estimated that 20,000–24,000 different mRNA sequences are present in maize pollen. A recent microarray analysis of the pollen transcriptome in Arabidopsis thaliana supports the idea of reduced complexity compared to the sporophyte, but provides a lower estimate: at least 3500 genes are expressed in pollen, or 12% of the genome (HONYS and TWELL 2003 ). Of these pollen-transcribed genes, 39% are either pollen specific or preferentially expressed in pollen, indicating a significant difference between pollen and sporophytic transcriptomes. Nonetheless, this leaves >61% of pollen genes expressed in both generations. After accounting for mRNA expression levels, the microarray analyses demonstrated that, compared to the sporophyte, pollen preferentially expresses genes involved in cell wall metabolism, the cytoskeleton, and signaling, suggesting that these cellular components are particularly crucial for pollen function (HONYS and TWELL 2003 ).

Despite the expression of numerous genes in pollen, relatively few mutants and variant alleles of gametophytically acting genes have been identified, and even fewer have been molecularly isolated (FRANKLIN-TONG 2002 ; JOHNSON and PREUSS 2002 ). In maize, quantitative trait loci (QTL) for pollen competitive ability have been mapped to specific chromosomal regions, including some thought to affect grain germination and pollen tube growth rate (SARI-GORLA et al. 1995 ). Moreover, at least eight different loci, termed gametophytic factors (Ga), have been described in maize (NELSON 1994 ). The Ga alleles confer a competitive advantage on male gametophytes that carry them, compared to gametophytes that carry the corresponding gametophyte factor (ga) alleles. Some Ga loci (e.g., Ga1) require specific genotypes in the female sporophyte to allow expression of this competitive difference. In A. thaliana, screens for mutations that alter Mendelian segregation ratios by eliminating or reducing transmission of a linked marker through the pollen have identified a number of gametophytically important genes (e.g., LIMPET POLLEN, HOWDEN et al. 1998 ; the MAD genes, GRINI et al. 1999 ; and the TTD genes, PROCISSI et al. 2001 ). Visual screens for morphological and cytological defects in pollen have also been fruitful (e.g., SIDECAR POLLEN, CHEN and MCCORMICK 1996 ; GEMINI POLLEN, PARK et al. 1998 ), as have reverse genetics approaches (e.g., LAT52, MUSCHIETTI et al. 1994 ; AtPTEN1, GUPTA et al. 2002 ). However, the majority of these mutations affect early stages of pollen development, resulting in aberrant pollen grains, and thus are less likely to be involved in the later stages that have been linked to pollen competition in natural populations. A few of the A. thaliana mutations (e.g., Ttd41, PROCISSI et al. 2001 ) do not cause obvious defects in the pollen grain and are transmitted through the male at low frequencies, identifying loci that appear to affect competitiveness after pollen grain development.

One class of proteins with clear links to both signaling and the cytoskeleton at late stages of pollen development is the Rop GTPase, a plant-specific subfamily of the Rho GTPases (ZHENG and YANG 2000 ; YANG 2002 ). Evidence from dicot species indicates that Rop GTPase activity is crucial for pollen tube growth, as well as a number of other plant signaling pathways, in which these proteins function as molecular switches. At least two A. thaliana Rops (ROP1 and ROP5/At-RAC2) help regulate polar growth of the dicot pollen tube (LIN and YANG 1997 ; KOST et al. 1999 ; LI et al. 1999 ; FU et al. 2001 ). Tube growth in vitro is arrested or slowed dramatically following inhibition of Rop function by antibody microinjection or by transgenic overexpression of a dominant-negative Rop allele. Furthermore, a constitutively active ROP1 or ROP5/At-RAC2 induces depolarized expansion of pollen tubes, along with defects in the organization of F-actin at the pollen tube tip (KOST et al. 1999 ; LI et al. 1999 ; FU et al. 2001 ). Rop has also been linked to both the Ca²⁺ gradient originating at the pollen tube tip (LI et al. 1999 ) and tip-localized activity of phosphatidylinositol monophosphate kinase (KOST et al. 1999 ), which, in turn, may regulate the cytoskeleton or other cellular components that affect polar growth. However, because no mutant alleles for these gametophytically acting ROP genes have been reported, the in vivo effects of altering specific Rop gene activity in pollen function have not been characterized. In addition, although Rop has been linked to disease responses in rice (KAWASAKI et al. 1999 ; ONO et al. 2001 ), information regarding Rop function in monocot development is limited.

There are currently nine known maize rops (rop1–9), which can be divided into three phylogenetically distinct groups of three genes each (CHRISTENSEN et al. 2003 ). There is significant spatial and temporal overlap of the nine rop transcripts, with high levels of all nine in sporophytic tissues associated with active cell division and expansion, suggesting that functional redundancy is likely among rops in the sporophyte. However, only a subset of rops (rop2, rop8, and rop9) is highly expressed in mature pollen (CHRISTENSEN et al. 2003 ). Thus, the likelihood of functional redundancy in the male gametophyte is reduced. In addition, rop2 (previously referred to as racB in HASSANAIN et al. 2000 ) and rop9 are in the same phylogenetic group as Arabidopsis ROP1 and ROP5, which have been implicated in the regulation of pollen tube growth (KOST et al. 1999 ; LI et al. 1999 ; FU et al. 2001 ). However, rop2 and rop9 are duplicate genes, showing 97% nucleotide and 99% amino acid identity, and could be genetically redundant in the male gametophyte. In fact, the two proteins differ by only a single conservative amino acid change that is unlikely to cause functional differences between them (Fig 1). Furthermore, the untranslated regions (UTRs) of the two genes are also highly conserved, showing 78% identity across 134 bp in the 5' UTRs, including 97% identity in the 39 bp immediately upstream of the start codon and 92% identity across the 400 bp of the 3' UTRs (data not shown).

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Maize ROP2 and ROP9 are nearly identical. The amino acid sequence for ROP2 is shown; identical amino acids in ROP9 are dots. The single amino acid difference (valine vs. alanine) between the two proteins (solid background) is in the carboxy-terminal domain involved in targeting small GTPases to the plasma membrane (YANG 2002 ). Mutational analysis of the related mammalian GTPase K-ras4B in this region has shown that it can still be successfully targeted to the plasma membrane despite major changes, provided that the changes avoid disrupting the region's polybasic character (ROY et al. 2000 ).

To investigate the in vivo function of maize rops, we adopted a genetic approach, isolating Mutator (Mu) transposon insertions in five maize rops and testing these mutations for effects on sporophytic and gametophytic development. We found that none of the 16 isolated mutations (including 5 in rop2) had obvious effects on the sporophyte; however, three mutant alleles of rop2 were transmitted at reduced frequencies through the male gametophyte. Further work showed that the wild-type rop2 pollen had a competitive advantage over mutant pollen, indicating that the ROP2 protein has an important role in male gametophyte function, despite the expression of the ROP9-mRNA in this same cell type. Thus, the hypothesized redundancy between the two nearly identical genes proved unfounded, as rop2 provides an important selective advantage in maize, an outcrossing species in which the male gametophyte would be exposed to competitive conditions in any open-pollinated population.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material and genetic methods:
The Trait Utility System for Corn (TUSC) methodology (BENSEN et al. 1995 ; MENA et al. 1996 ) was used in collaboration with Pioneer Hi-Bred International to obtain mutations in the rop2, rop3, rop4, rop6, and rop7 genes. Briefly, gene-specific primers (GSPs; Table 1) for each rop were used with a primer to the Mu transposon terminal inverted repeat (TIR) to screen a large population of mutagenized individuals for Mu insertions in each rop gene. To find heritable alleles, self-crossed F₂ progeny from PCR-positive F₁ parents were screened for the presence of a segregating GSP-Mu PCR product; these alleles are referred to as rop::Mu alleles. Amplified GSP-Mu fragments from heritable alleles were either directly sequenced or cloned into pPCR-Script Amp SK(+) using the manufacturer's protocol (Stratagene, La Jolla, CA) and then sequenced to confirm insertion into the gene of interest and to define the insertion site.

We generated Mu-inactive families carrying the rop::Mu alleles in either a W22 or a A188 inbred background. The rop::Mu alleles were first crossed from their initial Mu-active background to a bz1 sh1 stock and then to a Mu-inactive bz1-mum9 line. Mu-inactive progeny were selected by choosing bronze, unspotted kernels that were testcrossed to bz1 sh1 to confirm inactivity. The rop::Mu alleles were then introgressed into the two inbred lines. This introgression protocol aimed to remove genetic heterogeneity in the rop::Mu families, including any extraneous Mu insertions present in the original TUSC lines.

The wx1-marked reciprocal translocation (stock wx13A, T4-9b; breakpoints 9L.29; 4L.90) was obtained from the Maize Genetics Cooperation Stock Center (Urbana, IL). DNA derived from the IBM mapping population (SHAROPOVA et al. 2002 ), provided by the Maize Mapping Project (http://www.maizemap.org), was used to place rop2 on a chromosome arm by PCR genotyping.

PCR genotyping:
To determine the genotype of plants harboring the rop::Mu alleles, leaf DNA was extracted using either a rapid prep method (http://www.agron.missouri.edu/mnl/77/57vejlupkova.html) or a modified high-throughput method (PARIS and CARTER 2000 ) on a Matrix Mill (Harvester Technology; http://www.agron.missouri.edu/mnl/77/58vejlupkova.html). DNA preps from individual plants were subjected to PCR using a Mu TIR primer and rop GSPs. In most genotyping reactions, three primers (e.g., a rop2-forward, a rop2-reverse, and a Mu-TIR primer) were used; this multiplex reaction allowed discrimination among wild-type homozygotes, heterozygotes, and mutant homozygotes. Different rop GSPs were used depending on which allele was being evaluated (rop2 primers are listed in Table 1). The GenBank accession number for the rop2 genomic sequence is AY163379.

Analysis of gene expression by multiplex RT-PCR and quantitative real-time RT-PCR:
Pollen from at least two plants of each analyzed genotype was collected for 1–3 hr at anthesis, and pollen of a given genotype was pooled. Fresh pollen was ground into a paste using a mortar and pestle coated with Trizol (Invitrogen Life Technologies, San Diego). As the pollen was ground, 200-µl aliquots of Trizol, up to a total of 2 ml, were added (WEN and CHASE 1999 ). Total RNA was extracted from the homogenate according to the manufacturer's instructions for RNA with high polysaccharide content. The concentration of total RNA was determined spectrophotometrically, and the quality of RNA was assessed by running 5 µg of total RNA on a formaldehyde gel. Samples of total RNA were treated with DNaseI (RQ1, Promega, Madison, WI), and complementary DNA was synthesized with oligo(dT) primers using the Super Script First-Strand Synthesis system (Invitrogen Life Technologies) according to the manufacturer's instructions.

The ROP2-mRNA from wild-type (W22) and rop2::Mu plants was analyzed by RT-PCR using rop2 GSPs at both the 5' and 3' ends of the transcript. A primer pair to a widely expressed Elongation Factor1- (EF1-) gene (FERNANDES et al. 2002 ; PlantGDB ZMtuc03-04-07.21400) was used as an internal control (Table 1). RNA from the rop2-m3 allele was not analyzed because the maize line that harbored this allele did not efficiently shed viable pollen.

Real-time RT-PCR was performed on an ABI Prism 7700 sequence detection system (Central Services Laboratory, Oregon State University) using the ABI SYBR green PCR master mix kit (Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Primers for specific amplification of each cDNA were designed using the Primer Express software (Applied Biosystems) on the basis of parameters suggested for use with the ABI Prism 7700 and corresponding to regions near the 3' end of each cDNA (Table 1). Transcript levels of ROP-mRNA in pollen RNA samples were measured relative to transcript levels of PROFILIN1-mRNA (GenBank X73279; STAIGER et al. 1993 ) using the standard curve method for the ROP2 transcript and the Ct method for the ROP9 transcript (user bulletin no. 2, ABI PRISM 7700 sequence detection system). Each sample was measured three times, each time in triplicate, to obtain the relative transcript abundance. For comparison, the ROP2- and ROP9-mRNA expression values were then normalized relative to the wild-type sample, which was defined as 1 (user bulletin no. 2, ABI PRISM 7700 sequence detection system). PCR reactions were performed in triplicate in 25 µl using 500 nM each of forward and reverse primers, 1x SYBR green master mix (Applied Biosystems), and 2 µl of template in MicroAmp 96-well plates covered with optical caps (Applied Biosystems). Thermocycling conditions were as follows: 50°, 2 min; 95°, 10 min (1 cycle); 95°, 15 sec; 60°, 1 min (40 cycles); followed by an additional cycle required for melt analysis of 95°, 15 sec; 60°–95°, 20 min ramp; 95°, 15 sec (1 cycle). The melting curve was analyzed using the ABI PRISM dissociation analysis software (Applied Biosystems) to confirm the presence of specific products and determine whether the reaction produced any nonspecific products. Nonspecific amplification was not detected in any of the experiments performed.

In vivo pollen competition:
Pollen was collected at anthesis from bz1-marked homozygous rop2-m1 and rop2-m5 plants and bz1-marked homozygous wild-type (rop2+/rop2+) sibling plants for use as controls. Different lines of maize are known to produce pollen with differences in their competitive abilities (OTTAVIANO et al. 1988 ; SARI-GORLA et al. 1995 ), and thus wild-type rop2+ pollen from sibling plants was a necessary control with which to compare each type of rop2::Mu pollen. The bz1 seed phenotype was conferred by the bz1-mum9 (Mu-inactive) and/or bz1-sh1-x2 alleles segregating in these lines. To reduce potential differences due to genetic variation among individual plants, pollen collected from at least three different plants of each of the four rop2 genotypes was pooled to produce each pollen source. Competitor pollen from six W22 inbred plants (R1-scm2/R1-scm2; C1/C1; Bz1/Bz1) was also collected and pooled. A total of 0.5 ml of W22 competitor pollen was placed in a shoot bag with the same quantity of one of the bz1-marked rop2 pollen sources, producing four mixture types: W22/rop2-m1, its comparator W22/rop2+, W22/rop2-m5, and its comparator W22/rop2+. Each mixture type was crossed to three different bz1-sh1-x2/bz1-sh1-x2 (bronze, shrunken) tester ears. Recessive r1 and c1 alleles (colorless aleurone) segregated in both the rop2::Mu stocks and the bronze, shrunken tester stock; thus, seeds fertilized by a bz1-marked pollen population could produce either bronze or colorless kernels, whereas seeds fertilized by W22 produced only purple (full color) kernels. The sum of bronze (bz1/bz1) and colorless (r1/r1 or c1/c1) kernels was compared to the number of purple (Bz1/bz1; R1-scm2; C1) kernels on each ear to determine the competitive ability of each bz1-carrying pollen type relative to W22 (Bz1) pollen.

Pollen morphology:
Pollen from newly exerted anthers was placed on a microscope slide and stained with 0.25 µg/ml 4', 6-diamidino-2-phenylindole (DAPI). Microscopy was performed on a Zeiss Axiovert microscope with differential interference contrast optics and a UV filter set. Digital images were acquired using a SPOT CCD camera and software (Diagnostic Instruments, Sterling Heights, MI).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutator transposon insertions into rop2 are associated with a heritable, male-specific transmission defect:
We took a genetic approach to investigate the role of rop2 in pollen function and development in maize. A PCR-based screening technique (BENSEN et al. 1995 ; MENA et al. 1996 ) was used to isolate five independent Mu insertions into the rop2 gene: rop2-m1, rop2-m2, rop2-m3, rop2-m4, and rop2-m5. These alleles are referred to collectively as the rop2::Mu alleles. The rop2-m1 insertion was located in the 5' UTR, 26 bp upstream of the start codon (ATG), whereas the other four Mu insertions, rop2-m2, rop2-m3, rop2-m4, and rop2-m5, were located in the first intron 343, 368, 389, and 591 bp, respectively, downstream of the start codon (Fig 2). On the basis of the sequence in each allele of 39 bp of the Mu transposon terminal inverted repeats, the rop2-m2 and rop2-m5 alleles contained a MuDR element, rop2-m1 and rop2-m3 contained a Mu1 element, and rop2-m4 contained a Mu8 element.

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Isolation and identification of rop2::Mu alleles. (A) Genomic map of rop2 with Mu transposon insertion sites. The 5' UTR is shown as a thick solid line, and the coding regions of the first two exons are boxed. Classes of Mu transposons (not shown to scale) are identified by their fill patterns: cross-hatched, MuDR (5 kb); solid, Mu1; striped, Mu8 (both 1.4 kb; LISCH 2002 ). (B) PCR reactions using a Mu terminal inverted repeat primer and the gene-specific rop2 primer R8 (A) confirms the Mu insertion sites. (C) Portions of the genomic sequence for wild-type rop2 (GenBank accession no. AY163379) with the locations of the Mu insertions for rop2-m1, rop2-m2, rop2-m3, rop2-m4, and rop2-m5. The Mu insertion sites are numbered relative to the 5' end of the start codon (+1). The nine nucleotides of the wild-type rop2 sequence that were duplicated upon Mu insertion (in boldface type) are shown once; in the mutant alleles, the duplicated sequences are adjacent to, and on both sides of, each Mu insertion.

Initially, we noted that self-pollination of rop2::Mu heterozygotes resulted in full ears, but for three alleles (rop2-m1, -m2, and -m5), it produced fewer-than-expected mutant progeny, as determined by PCR genotyping. For PCR genotyping, DNA samples were prepared from each progeny plant, and a PCR reaction using two rop2-specific primers and a Mu primer allowed us to distinguish wild-type, rop2::Mu heterozygous, and rop2::Mu homozygous plants (as in Fig 3A). The initial self-pollinated families had lower-than-expected frequencies of both mutant heterozygotes and homozygotes. However, plants homozygous for each of the five rop2::Mu alleles were recovered, and none had any obvious defects, nor were they notably less vigorous than their wild-type and heterozygous siblings (data not shown).

View larger version (66K): [in this window] [in a new window] [Download PPT slide]   Figure 3. PCR genotyping identifies wild-type and rop2::Mu heterozygous plants. Heterozygous rop2-m1 mutant plants were used as either the female (A) or the male (B) in reciprocal crosses with a wild-type tester. Genotypes of the progeny (12 shown here) were ascertained by PCR using a Mu terminal inverted repeat primer and the rop2 R6 and F4 primers (Fig 1A). PCR products from plants heterozygous for the rop2-m1 allele produce three bands, two mutant (arrows), and one wild type (asterisk), whereas wild-type plants produce only a single band (asterisk). Mutant homozygotes produce only the two mutant bands (data not shown). The faint upper band at 500 bp in rop2-m1 heterozygotes is a PCR artifact, as confirmed by sequencing.

To determine whether the paucity of rop2::Mu genotypes was due to a defect in either gametophyte function (reduced transmission) or sporophyte viability (low-penetrance homozygous lethality), we checked transmission frequencies of the rop2::Mu alleles to the progeny in crosses using wild-type tester plants and rop2::Mu heterozygotes as either male or female. Progeny from each cross were genotyped by PCR, using the wild-type product as an internal control for the PCR reaction (Fig 3). Transmission of rop2::Mu alleles through the female and male gametophyte was analyzed over four generations (Table 2 and Table 3). Each line in Table 2 and Table 3 represents genotyped progeny from an independent cross; only data from families with at least 16 genotyped individuals are reported.

Three of the five independent rop2::Mu alleles (rop2-m1, -m2, and -m5) were associated with a heritable male-specific transmission defect, appearing in the progeny in numbers significantly less than the expected Mendelian ratio. No heritable defects in transmission through the female were associated with any of the five mutations, and neither rop2-m3 nor -m4 was associated with a male transmission defect. In the entire data set, the percentage of transmission through the male for the defective alleles ranged from 5 to 24% for rop2-m1, 5 to 53% for rop2-m2, and 6 to 33% for rop2-m5. Although rop2-m2 did not show a transmission defect in the first generation and in one of the second-generation crosses, it has remained associated with a defect in all subsequent generations. The anomalous results in the initial crosses could have been due to either genetic background effects or Mutator activity-related phenotypic suppression (MARTIENSSEN et al. 1989 ). The data were collected from populations produced by crossing in two distinct environmental conditions: field and greenhouse. Thus, the defect appears relatively constant in different environmental settings.

The low-frequency transmission of the rop2::Mu alleles through the male could have be due to recombination of rop2::Mu away from a second, linked mutation that was the actual cause of the transmission defect or high-frequency reversion to wild type of such a second mutation. However, the data set included two crosses each for rop2-m1 and rop2-m2, in which the mutant allele was transmitted through the male and, in the subsequent generation, was still associated with a transmission defect. These data argue that, at least for these two mutations, transmission of the rop2::Mu allele through the male did not remove its association with the defect, and that neither the recombination nor the reversion hypothesis was a likely explanation for the low-frequency male transmission of rop2::Mu. Rather, the rop2::Mu-associated defect appeared incompletely penetrant or incompletely expressed.

A linked marker was used to confirm that the reduced transmission phenotype was indeed associated with the long arm of chromosome 4, where rop2 was located. We mapped the rop2 gene to 1.8 cM proximal to umc1109 (bin 4.10) on the IBM genetic map (SHAROPOVA et al. 2002 ; data not shown). A reciprocal translocation between chromosome 4 and chromosome 9 (RT 4-9b) marked with the waxy endosperm (wx) seed phenotype was used to establish linkage of the wild-type Wx marker to the rop2::Mu alleles. Plants in which Wx was linked to either rop2-m1 or rop2-m5 displayed reduced transmission of the Wx marker through the male gametophyte as compared to wild-type rop2 (Table 4). These results were consistent with the rop2::Mu transmission data (Table 2), supporting the hypothesis that mutation of rop2 causes a male-specific transmission defect.

The rop2::Mu alleles can be placed in an allelic series:
We used a pollen competition assay to investigate the relative strengths of the rop2::Mu alleles. The mutant alleles were placed under competition as trans-heterozygotes and used as pollen parents in outcrosses to wild-type testers (Table 5). The relative transmission frequency of each rop2::Mu allele was used to determine the severity of the male-specific transmission defect. The results suggested that the rop2::Mu alleles represented an allelic series with a proposed order of severity from strongest to weakest: -m1 > -m2 > -m5 > -m4 (equivalent to wild type). It appeared that the rop2-m1 had the most adverse effect on rop2 activity, perhaps because the transposon was inserted near the translation start site.

Other maize rop::Mu mutants did not display a similar male-specific transmission defect. We isolated mutants for several other members of the rop family: rop3, rop4, rop6, and rop7 (Fig 4). In crosses using heterozygous rop::Mu plants as males to wild-type testers, transmission of all tested rop3::Mu, rop4::Mu, rop6::Mu, and rop7::Mu alleles was not significantly different from the expected 1:1 frequencies (Table 6). The alleles selected for these tests were primarily exon insertions (rop3, rop6, and rop7), and one of the rop4 alleles (rop4-m1) was an insertion in the 5' UTR. Thus, the male-specific transmission defect observed with the rop2::Mu alleles appeared specific to rop2. This correlated with the high level of ROP2-mRNA found in mature pollen, compared to the low or undetectable mature pollen mRNA levels associated with the other four rops (CHRISTENSEN et al. 2003 ). Mutant alleles for the other two rops expressed at high levels in mature pollen (rop8 and rop9) were not available for analysis.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Mutator transposon insertions in other maize rops do not affect transmission. Partial genomic maps of other maize rops, Mu insertion sites, and allele designations are shown. UTRs are represented by thick black lines and exons are boxed.

The male-specific transmission defect correlates with reduced and aberrant ROP2 transcripts:
Three of the rop2::Mu alleles, rop2-m1, rop2-m2, and rop2-m5, displayed the transmission defect phenotype. The rop2-m1 allele contained a Mu element in the first exon, whereas the other two alleles, rop2-m2 and rop2-m5, contained a MuDR-related element in the first intron (Fig 5A). MuDR is the autonomous element of the Mu transposon system and is 5 kb in length (LISCH 2002 ). In contrast, the rop2-m3 and -m4 alleles, which were not associated with a transmission defect, contained Mu1- and Mu8-related elements (1.4 kb in length) in the first intron. It seemed likely that, if the intron insertions caused a reduction in rop2 function, they would be associated with either aberrant or reduced transcript levels, perhaps due to splicing defects.

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Figure 5. RT-PCR demonstrates aberrant and reduced ROP2-mRNA in pollen from mutants with a transmission defect. (A) Genomic map of rop2 showing the Mu transposon insertion sites and the primers (arrows) used for RT-PCR. UTRs are represented by thick solid lines and exons are boxed. (B–D) All RT-PCR reactions included a rop2-specific primer set (arrows); plasmids (pl.) containing either a rop2 or a rop9 cDNA were used as control templates to demonstrate rop2 primer specificity and expected product sizes. An EF-1 primer set (asterisk) was included in each reaction as an internal control; the rop2-m5 sample appears to have a polymorphic EF-1 allele, resulting in a consistently larger product. (B) RT-PCR using a Mu primer and the rop2 R1 primer indicate that the ROP2-m1-mRNA contains Mu sequences (arrow). (C and D) RT-PCR using the rop2 F1 and R1 primer set (C) and the rop2 F2 and R2 primer set (D) demonstrate that a product the same size as that from the wild-type ROP2 transcript is present in pollen from rop2-m1, -m2, -m4, and -m5 homozygous mutants.

RT-PCR was used to investigate the ROP2 transcript in pollen from wild-type and homozygous mutant plants for four of the rop2::Mu alleles: rop2-m1, -m2, -m4, and -m5. The specificity provided by PCR was necessary to assay expression from rop2 due to the presence in the maize genome of rop9, an apparent duplicate of rop2 with high sequence identity at both the nucleotide and amino acid level with rop2 (Fig 1). RT-PCR using rop2 primers at both the 5' and 3' end of the gene amplified wild-type-sized bands in plants homozygous for all the rop2::Mu alleles, suggesting that correct splicing can occur in transcripts derived from each allele. However, the level of ROP2 transcript appeared to be reduced in the rop2-m1, rop2-m2, and rop2-m5 pollen, as compared to wild type (Fig 5C and Fig D). To determine if Mu element sequences were present in mature ROP2 transcripts, RT-PCR was performed on samples using a rop2 primer and a Mu inverted repeat primer (Fig 5B). In only one rop2::Mu allele, rop2-m1, was a band amplified, indicating the presence of Mu in mature ROP2-m1-mRNA. Sequencing of the amplified band confirmed that the Mu inverted repeat was present at the site corresponding to the DNA insertion site.

ROP2 transcript levels revealed a strict correlation between the rop2::Mu alleles that displayed a transmission defect and reduced production of ROP2-mRNA (Fig 6). Quantitative real-time RT-PCR using profilin1 (STAIGER et al. 1993 ) as an internal control was used to accurately measure transcript levels from both rop2 and the closely related rop9. In pollen from the rop2-m1, rop2-m2, and rop2-m5 mutants, the results indicated a 20-, 29-, and 16-fold reduction in ROP2-mRNA levels, respectively, as compared to the wild type. Conversely, the ROP2-mRNA levels in pollen collected from the rop2-m4 mutant, which was not associated with any transmission defect, did not show a significant reduction as compared to wild type. For all samples tested, changes in ROP9-mRNA levels ranged from a 1.6- to a 3.5-fold increase as compared to the wild type. The increase in ROP9 transcript levels, however, did not correlate with the observed transmission phenotype and may be due to differences in genetic background among the samples tested. To confirm that the dramatic reduction in ROP2-mRNA levels was not solely the result of genetic background differences among the analyzed samples, quantitative measurements were performed on a population segregating for rop2-m5 homozygotes, heterozygotes, and wild types. In these samples, a 6.3-fold reduction in ROP2-mRNA transcript in rop2-m5 homozygotes was observed, compared to wild-type siblings (data not shown). Moreover, rop2-m5 heterozygotes displayed a reduction in ROP2-mRNA to levels approximately halfway between those of the wild-type and homozygous mutants. These data strongly argue that the transmission defect is due to a mutation of the rop2 gene.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Quantitative real-time RT-PCR indicates that three rop2::Mu mutations considerably reduce ROP2-mRNA levels. Expression levels of ROP2- (solid bars) and ROP9-mRNA (open bars) relative to PROFILIN1-mRNA in pollen were measured via quantitative real-time RT-PCR (see MATERIALS AND METHODS). The expression values were then normalized relative to the wild-type level (i.e., wild type = 1) and displayed on a log-scale graph with bars corresponding to standard error. Each real-time measurement was repeated three times in triplicate to determine transcript abundance. Similar results were obtained from a second independent set of RNA isolations and measurements.

The rop2 mutation affects the competitive ability of the male gametophyte:
Crosses in which homozygous rop2::Mu plants were used as male parents produced full seed set and thus, in the homozygous state, the mutations did not notably affect pollen function (Fig 7A). This result suggested that the transmission defect was the result of a competitive disadvantage for rop2::Mu pollen in the presence of wild-type pollen, i.e., when collected from a heterozygote. One possibility was that rop2::Mu pollen expressed an early defect in pollen development such that mutant pollen was less likely than wild-type pollen to be shed and thus was already at a numerical disadvantage when placed on ear silks for pollination. Alternatively, rop2::Mu pollen could be at a disadvantage in the late stages of gametophytic development, e.g., during pollen tube growth. To distinguish between these possibilities, we used a pollen-mixing experiment (OTTAVIANO et al. 1988 ) to reconstitute a competitive environment for rop2::Mu pollen using known quantities of both mutant and wild-type pollen.

View larger version (161K): [in this window] [in a new window] [Download PPT slide]   Figure 7. The male-specific transmission defect is due to competition with wild-type pollen. (A) A rop2-m1 homozygous plant that was self-pollinated produced a full seed set. Self-crosses and outcrosses of homozygous rop2-m2, rop2-m4, and rop2-m5 plants gave similar results (data not shown). (B and C) A homozygous bronze shrunken tester (bz1-sh1-x2/bz1-sh1-x2) was pollinated with a 1:1 mixture of W22 inbred pollen and marked with the wild-type (Bz1+) purple allele and pollen from either a rop2-m1 homozygote (B) or a wild-type sibling homozygote (C), also homozygous for a bronze1 allele.

For the mixing experiment, three different maize lines were used as pollen sources: a rop2-m1-segregating population and a rop2-m5-segregating population, both homozygous for the recessive bronze1 seed marker (either bz1-mum9 or bz1-sh1-x2; see MATERIALS AND METHODS), and a W22 wild-type inbred line homozygous for wild-type Bz1+, as well as all other alleles necessary for purple seed color. Pollen was collected from two sets of sibling plants that were either homozygous rop2::Mu (rop2-m1 or rop2-m5) or homozygous rop2+ (control pollen). Each of these pollen types was mixed in equal quantities with pollen from the W22 wild-type inbred line. Thus, each of four different pollen mixtures consisted of equal parts Bz1+-marked W22 pollen and either bz1-marked rop2::Mu pollen or bz1-marked rop2+ (control) pollen. Females homozygous for a deletion of bz1 and the tightly linked sh1 gene (bronze, shrunken seeds) were pollinated with each pollen mixture (three ears for each mixture type), and transmission of each rop2 allele was monitored by comparing the number of purple progeny, fertilized by W22 pollen, to the number of nonpurple (bronze and colorless; see MATERIALS AND METHODS) progeny, fertilized by rop2::Mu or control rop2+ pollen (Fig 7B and Fig C).

The transmission frequency of the rop2::Mu allele, relative to the competitor W22 pollen, was compared to the transmission frequency of the rop2+ allele from sibling plants, relative to the same W22 competitor pollen (Table 7). Each line in Table 7 represents data collected from a separate ear. Transmission of the rop2-m1 allele ranged from 8 to 12%, compared to a 74–76% transmission rate for its rop2+ sibling. A reduction in transmission frequency was also observed with the rop2-m5 allele, which showed 1–7% transmission compared to a transmission rate of 19–21% for its rop2+ sibling. Because different maize lines produce pollen with differences in their competitive ability (OTTAVIANO et al. 1988 ; SARI-GORLA et al. 1995 ), we did not expect to see a 1:1 ratio of purple to nonpurple progeny with the W22/rop2+ control mixtures. Rather, our data indicated that, due to factors in the genetic background, the rop2-m1-segregating line produced wild-type pollen that was a better competitor than the W22 pollen (only 25% transmission of the W22 marker from a 1:1 mixture), whereas the rop2-m5-segregating line produced wild-type pollen that was a worse competitor (80% transmission of the W22 marker from a 1:1 mixture). However, in both cases, the presence of either rop2::Mu allele significantly detracted from the ability of pollen to successfully fertilize an ovule, as shown by the differences in transmission frequency between the rop2::Mu pollen and the rop2+ control pollen, relative to W22 pollen. For both experiments, the ratios of rop2::Mu: W22 progeny were significantly different from the ratios of rop2+:W22 progeny (rop2-m1 population, ² = 841.65, P < 0.001; rop2-m5 population, ² = 37.36, P < 0.001). Thus, for both of the rop2::Mu alleles that were tested, the rop2::Mu allele transmitted at a lower frequency than the rop2+ allele did, consistent with the phenotype found in mutant heterozygotes. These results confirmed that the male transmission defect occurred when rop2::Mu pollen was exposed to competition from wild-type pollen and further indicated that the defect that caused the competitive disadvantage manifested itself after pollen shed.

We have started investigating the specific role of rop2 in male gametophyte development. Microscopic observation of pollen from plants homozygous for rop2-m1, as compared to pollen from wild-type sibling plants, failed to show any obvious morphological differences (Fig 8A and Fig B). DAPI staining demonstrated that pollen grains from both rop2-m1 and wild-type homozygotes were trinucleate (Fig 8C and Fig D). Moreover, in vitro germination assays have thus far failed to reveal altered pollen tube morphology or reduced germination frequency in mutant as compared to wild-type pollen (data not shown). These results suggest that rop2 acts at a later stage of gametophyte development to provide a competitive advantage.

View larger version (90K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Pollen from rop2-m1 homozygotes has a wild-type morphology. Images of wild-type (A and C) and rop2-m1 (B and D) pollen were taken using transmitted light (A and B) or fluorescence optics to show DAPI staining (C and D). In pollen grains of both genotypes, the vegetative nucleus (vn) and the sperm nuclei (sn) exhibit characteristic diffuse and condensed morphologies, respectively. p, pollen grain pore. Bars, 50 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A role for rop2 in the male gametophyte:
Our work provides the first functional evidence for a role for rop in monocot development. We have shown that the observed male-specific transmission defect phenotype is the direct result of the mutation of rop2. The mutant phenotype was heritably associated with three independent rop2::Mu alleles over four generations, and each of these three alleles was associated with a dramatic reduction in ROP2-mRNA levels in the pollen. Moreover, the rop2-m4 allele, which was not associated with a transmission defect, also displayed no differences from wild type in either ROP2-mRNA level or transcript size and thus served as a "negative control." In addition, a genetic marker linked to rop2::Mu also displayed reduced transmission through the male, and the Mu insertions in four other rop genes were not associated with a similar male-specific transmission defect. Together, these results provide genetic confirmation for the role of rop2 in the male gametophyte, thereby genetically defining for the first time the in vivo importance of a specific rop in male gametophyte function.

The collection of rop2::Mu alleles described in this work represents an allelic series. The rop2-m1, rop2-m2, and rop2-m5 alleles displayed a male-specific transmission defect, with rop2-m1 having the strongest phenotype. In contrast, the rop2-m3 and rop2-m4 alleles did not notably decrease transmission through the male gametophyte. Because these alleles can be ordered, the biological function affected by rop2 can presumably be altered in a quantitative or graded manner. However, since the rop2::Mu alleles originated in distinct genetic backgrounds, we cannot discount the possibility that the differences in relative transmissibility of the three defective rop2::Mu alleles are due to effects from linked modifier loci, and not from rop2 itself.

Quantitative real-time RT-PCR experiments, using rop2-specific primers to the 3' end of the gene, detected ROP2 transcript in the pollen of all of the rop2::Mu homozygous mutants tested, indicating that none of the mutations completely eliminate ROP2-mRNA; i.e., none are RNA null (at least in the male gametophyte). Moreover, RT-PCR experiments directed at both the 3' and 5' ends of the gene did not indicate any gross differences in ROP2-mRNA structure in the intron-insertion alleles (rop2-m2, -m4, and -m5). One attractive hypothesis to explain the phenotypic differences between the four alleles with insertions in the first intron is that a large MuDR-related element (rop2-m2, -m5) exerts a much stronger deleterious effect on splicing than a smaller element does (e.g., Mu1 and Mu8, in rop2-m3 and -m4, respectively). Our RT-PCR analyses are consistent with this idea, and previous work has shown that the shorter Mu elements can be spliced out of mature transcripts if inserted into introns (LUEHRSEN and WALBOT 1990 ; ORTIZ and STROMMER 1990 ). This contrasts with the Mu1 insertion in the rop2-m1 allele, which is in the first exon, just upstream of the initiation codon, and is not spliced out of mature ROP2-mRNA. Thus, the rop2-m1 insertion causes both a reduction in transcript level and a structural change that could hinder translation. However, we cannot currently determine whether the strong rop2-m1 transmission defect results from an inability to produce ROP2 protein.

The competition component:
The male-specific transmission defect described in this investigation was detected only in crosses involving a rop2::Mu heterozygote or in mixtures of homozygous rop2::Mu and wild-type pollen. When relieved of competition from wild-type pollen, homozygous rop2::Mu pollen was competent to produce a full seed set. Furthermore, there was no observable sporophytic phenotype in homozygous rop2::Mu plants, although ROP2-mRNA is widely expressed in the sporophyte (CHRISTENSEN et al. 2003 ). Thus, rop2::Mu pollen displays a dramatically reduced fitness in the male gametophyte only when in a competitive environment, which could be due to defects in pollen germination, pollen tube growth rate and/or direction, or fertilization. Thus, rop2 can be considered a ga, a gametophytically expressed gene that affects pollen competitive ability (NELSON 1994 ). However, in contrast to several other maize ga loci (e.g., ga1), no effect of female genotype on expression of the rop2::Mu phenotype has yet been detected (K. M. ARTHUR and J. E. FOWLER, unpublished observations).

The maize rop2::Mu alleles are particularly noteworthy because they exist in a species that is naturally outcrossing and would offer ample opportunity for competition during pollination in a field-grown population. Thus, one would expect the three rop2::Mu alleles that cause a substantial transmission defect to be rapidly eliminated from maize populations due to strong negative selection, despite the absence of a discernible sporophytic phenotype. Intriguingly, a strong QTL for pollen competitive ability has been mapped to the tip of chromosome 4 (SARI-GORLA et al. 1995 ), the same location as rop2. Given that the rop2::Mu alleles form an allelic series and that reduction of ROP2-mRNA levels can affect transmission efficiency, we speculate that variant alleles of rop2 may constitute at least part of this QTL, thereby affecting pollen competition in cultivated populations of maize. We now have the opportunity to investigate this possibility, as well as the interplay of molecular genotype, gametophytic competitive fitness, and its effects on sporophytic populations.

The duplicate gene pair, rop2 and rop9:
Maize, an ancient allotetraploid, exhibits extensive remnants of sequence duplication, including duplicate genes (GAUT et al. 2000 ; GAUT 2001 ). Duplicated genes can retain their original function, acquire mutations that alter protein function or gene regulation, or be lost due to mutation or epigenetic silencing (reviewed in WENDEL 2000 ). In maize, the rop2 and rop9 genes are duplicates, displaying 97% nucleotide and 99% amino acid identity. In addition, both genes are highly expressed in mature pollen and share other similarities in their developmental expression profiles (CHRISTENSEN et al. 2003 ). Hence, it seems likely that rop2 and rop9 share several functions in maize and are, at least in part, functionally redundant. The observation that mutation of rop2 causes a measurable mutant phenotype is thus somewhat surprising and raises some intriguing questions as to the functions of rop2 and rop9 in the male gametophyte.

It is unlikely that the protein functions encoded by this gene pair have diverged, given their nearly identical amino acid sequences (Fig 1). Furthermore, although there is a formal possibility that ROP2-mRNA and ROP9-mRNA are differentially translated, any difference is unlikely to be substantial, given the similarity in sequence between the two transcripts, including both the 5' and 3' UTRs. We envision two more plausible, alternative hypotheses to explain the phenotype associated with the mutation of rop2. In the first, the activities of rop2 and rop9 are essentially equivalent, and the male gametophyte is highly sensitive to levels of rop2/rop9 activity; i.e., there is a dosage effect. In this scenario, rop9 and any remnants of rop2 activity in the mutant gametophyte provide a basal level of Rop function that allows the male gametophyte to carry out all required steps in fertilization. Full rop2 activity, however, allows a male gametophyte to fertilize an ovule much more efficiently and thus to out-compete a rop2::Mu mutant. In the second, the two genes are under differential regulation during male gametophyte development. In this scenario, efficient fertilization would require full rop2 activity, perhaps due to transcriptional downregulation of rop9 at later stages of gametophyte development (e.g., during pollen tube growth). This example highlights the possibility that the intense selective pressures acting during male gametophyte development, particularly in outcrossing species such as maize with heightened competition among gametophytes, could be an unappreciated mechanism to preserve duplicate genes in plant genomes.

The developmental defect associated with rop2::Mu alleles:
Previous studies defining a role for Rop GTPases in the male gametophyte have primarily used overexpression of dominant-negative ROP isoforms (KOST et al. 1999 ; LI et al. 1999 ), which are likely to affect promiscuously the activities of multiple ROPs (and possibly non-ROP pathways as well). Our genetic characterization corroborates these earlier studies, confirming the importance of Rop in the male gametophyte. However, our study precisely targets a single gene by mutating it; thus, we circumvent concerns of promiscuity associated with the dominant-negative ROP mutants and show that a single Rop, ROP2, is required for efficient pollen function in vivo. Our data also strengthen the inference (CHRISTENSEN et al. 2003 ) that maize rop2 is a functional homolog of A. thaliana ROP1, ROP3, and ROP5, which are all expressed in pollen (LI et al. 1998 ).

Overexpression of dominant-negative AtROP5 (KOST et al. 1999 ) or dominant-negative AtROP1 (LI et al. 1999 ) dramatically inhibits pollen tube growth rates in vitro, suggesting a possible mechanism to reduce pollen competitive ability. Antisense AtROP1 pollen tubes also grow more slowly than wild-type pollen tubes in vitro, but only at low Ca²⁺ concentrations; at high Ca²⁺ concentrations, these pollen tubes actually grow more rapidly than wild-type pollen tubes (LI et al. 1999 ). Thus it is unclear how reducing the activity of this single ROP would affect pollen tubes in vivo, and the in vivo consequences of altering Rop activity in any of these A. thaliana lines have not yet been shown. We have demonstrated that rop2-m1 homozygotes produce pollen grains with no obvious mutant phenotype, as compared to wild-type sibling pollen grains. In particular, the early stages of male gametophytic development (e.g., mitotic division, formation of the sperm nuclei) appear to occur normally (Fig 8). Moreover, the observation that the transmission defect is expressed after mixing rop2::Mu pollen with wild-type pollen suggests that the defect is manifested after pollen is shed, e.g., during or after pollen tube growth. Thus, rop2 appears to act at a later stage than most of the molecularly identified mutations that affect the male gametophyte [e.g., GEMINI POLLEN/MOR1 (TWELL et al. 2002 ) and AtPTEN1 (GUPTA et al. 2002 ), which both produce aberrant pollen grains]. The rop2::Mu phenotype may be similar to that of the A. thaliana Ttd41 (T-DNA transmission defect) mutation, which is associated with a partial male-specific transmission defect and has no apparent abnormalities in vitro (PROCISSI et al. 2001 ); however, the gene(s) responsible for this lesion have not yet been identified molecularly. Although the dominant-negative ROP studies suggest that a defect in pollen tube growth rate is one possible cause of the rop2::Mu transmission defect, other possibilities, e.g., a defect in the mutant's ability to locate the female gametophyte or to deliver the sperm nuclei to the egg sac, have not been ruled out. Our rop2::Mu allelic series should provide a spectrum of mutant phenotypes to help dissect any role of rop2 at these later stages of male gametophyte function.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AF126053 [rop2 (racB) transcript], AY163377 (rop9 transcript), AY163379 (rop2 genomic sequence), and AY163378 (rop9 genomic sequence).

	ACKNOWLEDGMENTS

The authors thank M. Foss and C. Rivin for useful critiques of the manuscript. We also thank C. Gasser and Z. Yang for helpful advice and discussions and acknowledge C. Albright, M. Baker, T. Christensen, C. Neou, O. Owusu, and E. Pease for help in the initial confirmation of several of the rop::Mu alleles, and the Oregon State University Center for Gene Research and Biotechnology Central Services Lab for their assistance with sequencing. This work was supported by a grant from the National Science Foundation (IBN-0111078) to J.E.F.

Manuscript received July 3, 2003; Accepted for publication September 8, 2003.

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