LARGE-SCALE BIOLOGY

The molecular dialog between flowering plant reproductive partners defined by SNP-informed RNA-Sequencing Alexander R. Leydon1, Caleb Weinreb1, Elena Venable1, Anke Reinders2, John M. Ward2, Mark A. Johnson1 1Brown University Department of Molecular Biology, Cell Biology, and Biochemistry 60 Olive St. Providence, RI 02912 2University of Minnesota Department of Plant Biology 140 Gortner Laboratory 1479 Gortner Ave. St. Paul, MN 55108-6106 USA Short title: SNP-informed RNA-seq of pollination One-sentence summary: Pollen- and pistil-specific patterns of gene expression were defined during Arabidopsis reproduction using single nucleotide polymorphism-informed RNA-seq analysis. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Mark A. Johnson (mark_johnson_1@brown.edu) Abstract The molecular interactions between reproductive cells are critical for determining whether sexual reproduction between individuals results in fertilization and can result in barriers to interspecific hybridization. However, it is a challenge to define the complete molecular exchange between reproductive partners because parents contribute to a complex mixture of cells during reproduction. We unambiguously defined male- and female-specific patterns of gene expression during Arabidopsis reproduction using single nucleotide polymorphism-informed RNA-seq analysis. Importantly, we defined the repertoire of pollen tube-secreted proteins controlled by a group of MYB transcription factors that are required for sperm release from the pollen tube to the female gametes, a critical barrier to interspecific hybridization. Our work defines the pollen tube gene products that respond to the pistil and are required for reproductive success; moreover, we find that these genes are highly evolutionarily plastic both at the level of coding sequence and expression across Arabidopsis accessions.

Plant Cell Advance Publication. Published on April 11, 2017, doi:10.1105/tpc.16.00816

©2017 American Society of Plant Biologists. All Rights Reserved

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Introduction

Flowering plants have evolved a unique reproductive system that depends on a

series of interactions between pollen and pistil that ensure two sperm cells are delivered

to each ovule. A pair of genetically identical sperm develop within a pollen grain and are

transported within the cytoplasm of the pollen tube, a highly polar extension of the

pollen cell (Berger and Twell, 2011). The pollen tube penetrates and extends through

floral tissue and responds to guidance cues (Marton et al., 2005; Okuda et al., 2009;

Takeuchi and Higashiyama, 2012; Mizukami et al., 2016; Takeuchi and Higashiyama,

2016; Wang et al., 2016) that direct it into an ovule, where the pollen tube encounters a

synergid cell, one of two supportive cells that flank the egg. The pollen tube then bursts

just as one of the two synergids degenerates (Denninger et al., 2014; Ngo et al., 2014;

Leydon et al., 2015); pollen tube rupture propels the pair of sperm to a position where

they contact the egg and central cell (Hamamura et al., 2011; Ngo et al., 2014). Two

simultaneous, or nearly simultaneous, plasma membrane fusion events then occur;

each releases a sperm nucleus into either the egg or central cell cytoplasm (Denninger

et al., 2014; Hamamura et al., 2014) for nuclear fusion. Double fertilization is the

defining feature of flowering plants and initiates development of an embryo and a

supportive endosperm, the major components of seeds.

To understand the molecular mechanisms that mediate pollen tube delivery of

sperm and double fertilization, it is important to define the gene expression patterns of

the critical cells as they interact with each other. Genomics of individual cells and

tissues has provided key insights into the transcriptomes of pollen (Becker et al., 2003;

Honys and Twell, 2003; Pina et al., 2005), embryo sac (Yu et al., 2005; Johnston et al.,

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2007); (Jones-Rhoades et al., 2007; Steffen et al., 2007), pistil (Scutt et al., 2003; Tung

et al., 2005) (Swanson et al., 2005), sperm (Borges et al., 2008), and isolated egg and

central cells (Dresselhaus et al., 1994; Wuest et al., 2010). Furthermore, comparing the

transcriptomes of pollen tubes grown in vitro (Wang et al., 2008) with pollen tubes

collected after they had grown through a pistil explant (semi-in vivo condition, SIV, Qin

et al., 2009) identified dynamic regulation of pollen tube transcription during growth

through the pistil. However, none of these studies provide a comprehensive and

integrated analysis of the timing and relationships between pollen tube and pistil cell

types as they interact over the eight hours required for the pollen tube to reach an

ovule. Importantly, microarray profiling of pollinated pistils unveiled dynamic changes

over 8 hours of pollen tube growth in vivo (Boavida et al., 2011); however, in this study,

it was impossible to distinguish pollen tube from pistil gene expression.

In Arabidopsis, pollen tubes grow through a complex extracellular matrix and

form hundreds of cell-cell contacts en route to the female gametes within an ovule. It is

likely that numerous cell-cell recognition and signaling exchanges are required to

promote successful fertilization by appropriate pollen genotypes and to block exotic

pollen genotypes. For example, there is strong evidence that species-specific cell-cell

interactions are required for pollen tube guidance (Marton et al., 2012; Takeuchi and

Higashiyama, 2012, 2016; Wang et al., 2016) into the ovule and for pollen tube

reception resulting in sperm release (Williams et al., 1986; Escobar-Restrepo et al.,

2007; Leydon et al., 2014; Muller et al., 2016). How the pollen tube and pistil recognize

each other and transduce identity information into alterations in the transcriptomes of

critical cells is unknown. However, a group of three pollen tube-expressed MYB

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transcription factors have been shown to be induced by growth through the pistil and

are required for the ability of pollen tubes to burst and release their sperm upon arrival

at the female gametophyte (Leydon et al., 2013; Liang et al., 2013). These findings

suggest a complex network of transcriptional regulation critically important for cellular

interactions responsible for flowering plant reproduction.

We set out to identify sex-specific (pollen tube vs. pistil) gene expression

patterns during pollination when pollen tubes are enmeshed within the pistil tissue and

actively engaged in signaling processes required for fertility. We sought a method that

allowed us to define changes in pollen- and pistil-transcript abundance that was not

invasive, did not require an in vitro system, and did not use transgenic markers. A major

challenge for genomics has been to develop techniques that define the transcriptomes

of individual cells or cell types within complex tissues. This type of analysis was

pioneered in the Arabidopsis root using cell-type-specific marker lines that facilitated

cell-sorting followed by microarray analysis (Birnbaum et al., 2005). More recently,

expression of pollen tube-specific tagged ribosomal protein facilitated the biochemical

isolation of pollen tube transcripts associated with ribosomes (Lin et al., 2014).

We took advantage of the modular nature of pollen by applying pollen of one

Arabidopsis accession to the pistil of another. We reasoned that polymorphisms in

transcripts would allow us to distinguish RNA-sequencing (RNA-seq) reads derived from

the pollen tube from those expressed in the pistil. This approach was used to

successfully differentiate monoallelic from biallelic gene expression during embryo and

endosperm development (Autran et al., 2011; Gehring et al., 2011; Nodine and Bartel,

2012; Pignatta et al., 2014). More recently, this approach was applied to analyze

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patterns of gene expression associated with pollen–pistil compatibility in interspecific

crosses of tomato species (Pease et al., 2016). Using this approach, we have analyzed

pollen tube and pistil gene expression in crosses between Cape Verde Island-0 (Cvi)

pistils and Columbia-0 (Col) pollen. Interestingly, analysis of gene expression in hybrid

embryos revealed that the onset of paternal allele expression varied depending upon

the combination of parental accessions (Autran et al., 2011; Nodine and Bartel, 2012;

Del Toro-De Leon et al., 2014). Our application of this approach analyzes gene

expression in reproductive tissues before formation of any hybrid cells (embryo and

endosperm). Nonetheless, we were curious to determine whether pollen tube gene

expression was altered depending on the genotype of the pistil through which it grew.

Our study provides a comprehensive analysis of pollen tube and pistil gene

expression in their native context and has revealed transcriptional changes that occur

specifically in either the pistil or pollen tube component of a gene expression profile:

changes not identifiable by other methods. Furthermore, we applied this approach to

define gene expression controlled by pollen-tube MYB transcription factors that control

pollen tube reception signaling (Leydon et al., 2013; Liang et al., 2013) and identified a

wealth of small, secreted proteins that may be critical for signaling from the pollen tube

to the pistil. Our analysis revealed differences in sex-specific gene expression patterns

across accessions and prompted us to analyze the patterns of selection that have

shaped the evolution of gene expression during pollination. We found a higher rate of

polymorphisms in the coding sequence of pollen tube- and pistil-expressed genes

compared to the background rate and that a family of MYB-dependent, secreted,

cysteine-rich proteins is diversifying rapidly in Arabidopsis via expansion and positive

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selection. These findings are consistent with the hypothesis that MYB-dependent pollen

tube-expressed genes diversify rapidly and reinforce boundaries to interspecific

hybridization by generating a species-distinct molecular signature.

Results A SNP database to determine whether RNA-seq reads from pollinated pistils are

derived from the pollen or pistil parent. There are ~100–200 pollen tube cells and

thousands of pistil cells in a pollinated pistil, so we estimated ~1% of the pollinated pistil

transcriptome would be pollen tube-derived. To maximize the likelihood that we would

detect RNA-seq reads unambiguously defining pollen tube transcripts, we chose two

Arabidopsis accessions, Col and Cvi, with a high SNP frequency (approximately 1 SNP

every 200bp, Schmid et al., 2003; Nordborg et al., 2005). We created a custom

database of Cvi SNPs that had been validated by genome sequence (Gehring et al.,

2011; Nodine and Bartel, 2012; Pignatta et al., 2014). To capture the greatest number

of SNPs in expressed genes from Col and Cvi, we incorporated additional SNPs

identified from RNA-seq of unpollinated and self-pollinated Cvi pistils (eight hours after

hand self-pollination, Figure 1A). Variant analysis of unpollinated and self-pollinated Cvi

pistils added 15,995 new SNPs to the previously identified 576,722 Cvi SNPs (Pignatta

et al., 2014), resulting in a comprehensive variant list used in all subsequent analyses (1

SNP every 201 bp; Supplemental Data Set 1). As 36.2% of these SNPs occur in exons

(coding sequences and untranslated regions, UTRs), they are useful for determining the

parent of origin of individual RNA-seq reads from pollinated pistils (Figure 1B,

Supplemental Figure 1). Importantly, 75% of annotated Arabidopsis genes had at least

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one SNP in a transcribed gene region, providing the potential to quantify pollen tube

and pistil transcript abundance unambiguously (24,441/32,678 = 74.7%).

Eight percent of total mRNA within the pollinated pistil is derived from the pollen

tube. We used bioinformatic approaches previously developed to define allele-specific

gene expression in the developing embryo and endosperm (Gehring et al., 2011;

Pignatta et al., 2014) to determine whether RNA-seq reads contained SNPs and

whether reads with SNPs were derived from the pollen tube or the pistil (Supplemental

Figure 2 [analysis workflow], Materials and Methods). We utilized methods that count

reads mapping to a single genome location (HTseq) or that count reads mapping to

multiple locations (EasyRNASeq, Supplemental Figure 2, Supplemental Data Set 2,

Supplemental Data Set 3 [comparison of methods]). In pollinated pistils, 12% of 131

million singly-mapped reads analyzed contained SNPs (Figure 1C) and these reads

were distributed across the transcribed regions of genes similarly to reads without SNPs

(Supplemental Figure 3, Supplemental Data Set 2). Inclusion of reads that were

mapped to multiple locations did not significantly alter the proportion of reads with useful

polymorphisms (Supplemental Data Set 2); however, multiply-mapped reads facilitated

enhanced detection of members of gene families expressed in pollen. So, multiply-

mapped reads were utilized for subsequent analyses.

These data suggest that in a typical experiment, ~17 million reads with SNPs are

available to quantify patterns of transcript abundance in the pollen tube and pistil.

Importantly, we determined that ~60% of genes expressed in the pollinated pistil have at

least one SNP, allowing unambiguous determination of parent of origin (Figure 1) in

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crosses between Col and Cvi. This analysis shows that we will not be able to

unambiguously determine the parent of origin for all genes using Cvi pistils pollinated

with Col pollen. Further bioinformatic analysis of sequenced Arabidopsis accessions

showed that crosses between Col and Pu2-8 would provide the greatest number of

additional useful SNPs (~20% more genomic features, Supplemental Figure 4).

To determine the relative contributions of pollen tube and pistil to the pollinated

pistil transcriptome (Figure 1A), we analyzed the total number of reads from Cvi pistils

pollinated with Col pollen that contained SNPs (Figure 1C) and found that 92% of these

reads had the Cvi allele (pistil reads) while 8% had the Col allele (pollen tube reads,

Figure 1C). These data show that pollen tube RNA constitutes ~8% of the total RNA

extracted from a pollinated pistil. Moreover, a scatter plot comparing the number of pistil

reads (Cvi, y-axis) with pollen tube reads (Col, x-axis) for all genes with SNPs shows

that pistil transcripts are much more abundant for the vast majority of genes (y-axis is

10 fold greater than x-axis). This is not surprising given the abundance of pistil cells

compared with pollen tubes. However, genes with expression values along the x-axis

point to a significant fraction only expressed from the pollen tube and readily detected

using this method (Figure 1D). Analysis of these data defines groups of pollen-

expressed, and pistil-expressed genes.

Sex-specific transcript analysis reveals the parent of origin of genes expressed

during pollination. A scatter plot comparing the number of total reads/gene in

unpollinated pistils with pollinated pistils identifies genes with expression patterns that

changed upon pollination and highlights a group of genes that are only detected upon

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pollination (Figure 1E, points along x-axis). The simplest hypothesis is that these genes

are expressed in the pollen tube; however, it is possible that some of these genes are

pistil-expressed genes induced in response to pollen tube growth. To distinguish

between these possibilities, we color-coded the scatterplot as follows: pollen tube-

expressed genes are shaded blue (>10 average pollen reads), pistil-expressed reads

are shaded red (>10 average pistil reads), and genes expressed in both tissues (>10

average pollen and pistil reads) are assigned a color between red and blue based upon

the ratio of pistil to pollen reads (Figure 1E, Supplemental Data Set 3). Pollen gene

expression is primarily responsible for the group of genes that are highly induced during

pollination (Figure 1E, blue dots).

To define pistil genes that responded to pollination, we focused on reads

containing Cvi SNPs in unpollinated (Cvi) versus pollinated pistils (Cvi x Col,

Supplemental Figure 2). We utilized the DESeq2 software package (Love et al., 2014),

which uses read counts and estimates variance-mean dependence and tests differential

gene expression using the negative binomial distribution and does not normalize

expression to the length of genes within libraries. This feature of DESeq2 was

advantageous because we wanted to compare the expression of individual genes

across conditions using the subset of reads that included SNPs. While a majority of

pistil-expressed genes remain unchanged by pollination (Figure 1E, red dots along

diagonal), it is clear that a number of pistil-expressed genes (pistil reads only) are

differentially expressed in response to pollination; 476 genes are significantly below the

diagonal (induced by pollination) or above the diagonal (higher in unpollinated pistils,

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DESeq2, methods, Supplemental Data Set 4), and could play roles in modulating the

pistil environment to promote directed pollen tube growth.

To identify pollen-specific and pistil-specific genes, we normalized the number of

sex-specific reads over each gene to the number of SNPs/gene. Then, we applied a

two-fold enrichment criterion for pollen-specific genes: >20 Col reads/SNP/gene, and

<10 Cvi reads/SNP/gene (average of three replicates). The reciprocal criteria were used

to identify pistil-specific genes. Normalization by SNPs/gene accounts for the large

variation in the SNP frequency across genes (1 to >300, Supplemental Data Set 3).

These stringent thresholds identified 277 genes expressed specifically from the pollen

tube and 6,838 genes expressed specifically from the pistil (Figure 1F). The ~25 fold

higher number of pistil-specific genes is due, at least in part, to the vast excess of pistil

tissue.

To determine whether SNP-informed determination of sex-specific gene

expression identifies genes known to be expressed in the pollen tube or pistil, we

analyzed genes defined in published microarray data. We graphed read coverage over

genes and color-coded reads to indicate whether they contained a pollen- or pistil-

derived SNP (red, pistil SNP; blue, pollen SNP), or whether they had no SNP (gray,

Figure 2A–F). AT1G47530, AT2G13690, and AT5G46800 had been shown to be

abundant in pistils (Boavida et al., 2011), but absent or minimally detectable in pollen

grain and pollen tube microarray experiments (Becker et al., 2003; Honys and Twell,

2003; Pina et al., 2005; Qin et al., 2009). All of the reads with SNPs carried the Cvi

allele for these genes (red, Figure 2A-C), indicating that in pollinated pistils all of the

RNA transcribed from these genes is transcribed in pistil cells.

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AT1G29140, AT1G55560, and AT2G26450 were shown to be expressed in semi

in vivo (SIV) grown pollen tubes (Qin et al., 2009), but were absent in unpollinated pistils

(Boavida et al., 2011). We found that all of the reads with SNPs carried the Col allele for

these genes (blue, Figure 2D–F), indicating that in pollinated pistils all of the RNA

transcribed from these genes is transcribed in pollen tubes.

Finally, we took the 10 most highly expressed genes detected in microarray

analysis of pollinated pistils (Boavida et al., 2011) and graphed the distribution of reads

for SNP-containing genes (8/10 genes contained useful SNPs). Four representative

genes are shown (AT4G10340, AT4G35100, AT5G02380, and AT1G72260, Figure 2,

G–J). These genes highlight one of the chief advantages of SNP-informed RNA-seq

analysis of reproductive gene expression; they clearly show that the male and female

components of gene expression can be determined unambiguously for genes

expressed in both the pollen tube and the pistil.

SNP-informed RNA-seq correlates with previously defined reproductive tissue

transcriptomes. Comparison of sex-specific gene expression defined by SNP-

informed RNA-seq with previous microarray analysis of component cell and tissue types

indicates overall similarity, but also identifies genes missed in prior analyses. One of the

principal advantages of SNP-informed RNA-seq analysis of pollination is that the pollen

tube and pistil components of gene expression can be determined without invasive

procedures that potentially distort native expression patterns. Prior to this analysis, it

was necessary to piece together male and female gene expression patterns from

experiments that interrogated pollen (Honys and Twell, 2004; Wang et al., 2008; Qin et

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al., 2009) and pistil separately (Swanson et al., 2005; Boavida et al., 2011). We

performed a Spearman’s correlation analysis to test whether sex-specific

transcriptomes determined using SNP-informed RNA-seq correlate with those of

component cell and tissue types previously determined by microarray analysis (Figure

2K, Supplemental Figure 5). Correlations with microarray experiments were limited

because the lists of gene identifiers are not completely overlapping. Nonetheless, we

found that in Cvi x Col pollinations, pollen-expressed genes (reads containing Col SNP)

demonstrated a high correlation with pollen transcriptomes, especially SIV-grown pollen

tubes (Figure 2K, left column; dark red is the highest Pearson coefficient observed).

Conversely, genes from Cvi x Col pollinations identified as pistil-expressed (reads

containing Cvi SNP) demonstrated a high correlation with pistil transcriptomes,

especially pistils pollinated for 3.5 or 8 h (Figure 2K, right column). As expected, pistil-

expressed genes defined by SNP-informed RNA-seq did not correlate with previously

identified pollen development, tube growth (in vitro and SIV pollen tubes), or vegetative

tissue profiles (Figure 2K, right column). Therefore, SNP-informed RNA-seq analysis

provides a non-invasive, in vivo analysis of gene expression that occurs while the pollen

tube is growing through pistil tissue and thus provides a more biologically relevant view

of gene expression during pollen tube–pistil interactions.

We compared pollen tube-expressed genes defined by SNP-informed RNA-seq

analysis with lists of genes previously defined by microarray analysis of SIV-grown

pollen tubes (Qin et al., 2009), unpollinated and pollinated pistils (Boavida et al., 2011),

and by translating ribosome affinity purification of pollen tubes growing through pistil

tissue (Lin et al., 2014). All comparisons are reported in Supplemental Data Set 5. The

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SIV transcriptome included 7,275 expressed genes, whereas SNP-informed RNA seq

identified 4,375 genes with RNA-seq reads that included the Col SNP (pollen reads);

the overlap was 28% (2,540 genes). This discrepancy was not due to a lack of SNP

coverage: only 31 SIV genes were not identifiable because they lack a Col - Cvi SNP.

Conversely, we identified a much larger group of genes (4,704) with useful SNPs that

were not detected as pollen tube-expressed genes in vivo, but that were detected as

pollen tube-expressed genes using the SIV method. Some of these genes may have

been induced by the manipulations of the SIV method in which pollen tubes exit pistil

tissue and are exposed to air while growing on agar media (Qin et al., 2009). We also

identified 1,735 genes with clear evidence for pollen tube expression in the pollinated

pistil (>10 Col reads) that were not detected by the SIV method. These newly identified

pollen tube expressed genes include genes not present on the ATH1 microarray and

genes that require native interaction with pistil tissue for their expression.

We found substantial overlap between the 267 pollen tube-specific genes

identified by SNP-informed RNA-seq (Supplemental Data Set 5) and 328 previously

identified pollination-induced genes (Boavida et al., 2011, overlap = 38%, 123 in

common). This is not surprising given that both analyses began with RNA isolated from

intact pollinated pistils. By contrast, there was no overlap between the genes defined as

pollen tube-specific by SNP-informed RNA-seq and by translating ribosome affinity

purification (n=469, Lin et al., 2014, Supplemental Data Set 5). Comparison of all genes

identified as pollen expressed in our study (n=3,939) with the pollen tube-specific genes

identified by translating ribosome affinity purification identified a modest overlap of 69

genes. The higher degree of overlap between our method, and microarray analysis of

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SIV-grown pollen tubes (Qin et al., 2009), and pollinated pistils (Boavida et al., 2011) is

likely because these techniques begin with total RNA rather than RNA associated with a

tagged ribosomal subunit that must compete with endogenous ribosomal subunits (Lin

et al., 2014).

The sex-specific components of the pollination transcriptome comprise distinct

functional categories. We found that pollen tube-specific genes were enriched for

gene ontology (GO) terms suggesting functions in pectin modification (14-fold; p

value=2.04x10-8, all GO enrichment p-values were determined using the Benjamini

method), cell–cell signaling (25-fold; p-value=1.4x10-7), calcium-mediated signaling (14-

fold; p-value=1.23x10-6), and pollen tube growth (8-fold; p-value=5.5x10-4). Analysis of

GO cellular component annotations revealed 27.9% of transcripts encoded genes

predicted to enter the endomembrane system (n = 74, p-value= 2.72x10-15), which

includes nine members of the Rapid Alkalinization Factor (RALF) gene family (27-fold;

p-value=1.18x10-7, Supplemental Data Set 6), which have been found to participate in

extracellular signaling (Haruta et al., 2014; Du et al., 2016; Stegmann et al., 2017). On

the other hand, there were 476 pistil-expressed genes that were differentially regulated

upon pollination: 216 were up-regulated upon pollination, 259 decreased abundance

upon pollination (Supplemental Data Set 7). Secreted glycoproteins (n = 38, p-value=

7.25x10-12) and secreted cell wall localized proteins (n = 26, p-value= 6.31x10-9) both

increase upon pollination as well as a small but significant number of genes involved in

response to chitin (n=4, p-value= 0.001) and response to auxin (n=8, p-value= 0.005).

These enriched GO categories may indicate parallels between the pistil’s response to

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pollen tube growth and previously characterized biotic stress responses. By contrast,

there is widespread decrease in expression of cytochrome p450 (n = 12, p-value= 4x10-

6) and oxidative stress genes (n = 10, p-value= 4x10-4) upon pollination. Of the 476

pistil-expressed genes, 168 were found to be pistil-specific by our criteria, of which 46

are predicted to be secreted (Supplemental Data Set 7). These observed changes in

expression likely indicate a major shift in the transcriptional program of female tissue

during pollination.

SNP-informed RNA-seq analysis defines the role of MYB transcription factors in

regulating pollen tube gene expression and the pistil’s response to pollination.

Pollen tubes lacking three closely related MYB transcription factors (Stracke et al.,

2001), MYB97, MYB101, and MYB120, fail to release their cargo of two sperm upon

entering an ovule. Consequently, myb triple-mutant pollen tubes continue to grow within

the female gametophyte producing a coil of pollen tube that fails to fertilize female

gametes (Leydon et al., 2013; Liang et al., 2013). It was proposed that pollen tube

MYBs respond to the pistil environment and in turn activate the expression of genes

critical for pollen tube reception signaling (Leydon et al., 2013; Leydon et al., 2014), the

interaction between the male and female gametophyte that results in sperm release

(Kessler and Grossniklaus, 2011). This model was explored using microarray analysis

of triple-mutant pollen grains (Liang et al., 2013) or pollinated pistils (Leydon et al.,

2014). It is impossible to know the parent of origin of transcripts using microarray

analysis, so neither of these approaches were able to determine the pollen- and pistil-

expressed genes with altered transcript abundance in mutant compared to wild-type

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pollinations. Importantly, microarray analysis failed to identify differential expression of

the pollen component of a gene expressed in both the pollen and the pistil, because

changes in the pollen tube component were masked by the much larger pool of pistil

transcripts. SNP-informed RNA-seq provides an opportunity to determine the changes

in gene expression due to loss of critical transcriptional regulators of the pollen tube cell

as it interacts with the pistil tissue it has evolved to navigate.

First, we compared Cvi x Col pollinations to Cvi x myb triple-mutant (accession =

Col) pollinations and analyzed differential expression using all mapped reads (Figure

3A-B). This analysis is similar to the previous microarray analysis because it ignores the

parent-of-origin of transcripts. As expected, RNA-seq identified the majority of the

differentially expressed genes previously identified by microarray analysis of pollinated

pistils (~63%, 30/48, DEseq2, Leydon et al., 2013); however, the overlap with

microarray analysis of MYB-dependent gene expression in pollen grains was much

lower (16%, 4/25, DEseq2, Liang et al., 2013). This difference between the MYB

regulatory networks of the pollen grain and the pollinated pistil is consistent with our

previous observation that pollen tube MYBs are induced by growth through the pistil and

likely regulate unique targets as the pollen tube grows through pistil tissue (Leydon et

al., 2013). Importantly, RNA-seq analysis uncovered an additional 288 MYB-dependent

genes (318 total, DEseq2, Supplemental Figure 6, Supplemental Data Set 8, Figure

3B). Genes with significantly lower expression in myb triple-mutants were enriched in

several functional categories including transmembrane transporters, carbohydrate

active proteins, and secreted proteins, as was previously defined (Supplemental Data

Set 8, Leydon et al., 2013).

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Next, we implemented sex-specific read identification on RNA-seq data from

wild-type and mutant pollinations to define the patterns of differential expression

resulting from loss of MYB97, MYB101 and MYB120 in pollen tubes and the pistil. This

analysis identified 278 pollen-expressed genes and 34 pistil-expressed genes that are

differentially expressed in myb triple-mutant pollinations (Supplemental Data Set 9).

When the mutant versus wild type total read scatter plot (Figure 3B) is color-coded to

highlight genes with significant changes in the pollen tube (blue) or pistil (pink, Figure

3C) component of their expression profile, it becomes clear that the total abundance of

many potentially MYB-regulated transcripts does not change even though the pollen

component does (blue dots along diagonal). Interestingly, this analysis also defines a

small number of pistil-expressed genes that change significantly when the pistil is

pollinated by mutant compared to wild-type pollen tubes (pink dots, Figure 3C).

By comparing the differentially expressed genes identified using total reads

(ignoring parent-of-origin) with those determined following analysis of sex-specific

reads, we find that 218 differentially expressed genes were unmasked when differential

gene expression was determined for pollen and pistil reads independently (Figure 3C).

This finding shows that SNP-informed RNA-seq is successful at defining changes in the

pollen- and pistil-specific components of gene expression for those genes expressed in

both tissues and indicates that many genes are under regulatory control in the pollen

tube that is distinct from that of the pistil. However, since we used a different

bioinformatic procedure to initially align total reads than that used to process reads with

SNPs (Methods), we repeated this analysis and compared sex-specific reads with all

reads lacking a SNP; the result was similar to our previous analysis (Figure 3D). Many

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pollen tube-expressed genes were determined to be differentially expressed in myb

triple-mutants whether or not reads with SNPs were analyzed (Figure 3E dark blue

intersection, n=68); however, an additional 210 genes are identified by determining

whether pollen reads (Col SNP) are differentially abundant (p < 0.05, Wald test,

DESeq2) in mutant compared with wild type. Pollen tube-specific MYB-dependent

genes are comprised of several functional annotation categories, the most enriched

being transmembrane transporters (n=26, p-value = 4.46x10-5, Benjamani test,

Supplemental Data Set 9).

By contrast, only a few pistil-expressed genes (34 genes, Figure 3E) were found

to be differentially expressed in mutant compared with wild-type pollinations. Thirteen of

these were also identified upon analysis of pistil (Cvi) reads. This small, but interesting

gene set is enriched in chitin-responsive genes (n = 6, p-value = 9.08x10-5, Benjamani

test), and may represent pistil genes that respond to myb mutant pollen tubes, which

are perceived as different from wild type (Supplemental Data Set 9).

To illustrate the consequence of MYB loss-of-function on the pollen tube

component of gene expression, we plotted read coverage for two genes identified as

differentially regulated in myb triple-mutant pollen tubes (Figure 3F, Supplemental Data

Set 9). In wild-type pollinations (Figure 3F, top graph) the RNA-seq reads covering

SNPs comprise both pistil (Cvi, red) and pollen (Col, blue) reads, with a ten-fold greater

contribution from the pistil. In myb triple-mutant pollinations the pollen component (Col,

blue) disappears, yet the much larger pistil component is unchanged. This analysis

shows the utility of SNP-informed RNA-seq analysis of reproduction, which facilitates

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independent analysis of the pollen tube and pistil components of gene expression as

the pollen tube interacts with the pistil.

Genetic reporter constructs corroborate SNP-informed RNA-seq analysis.

We produced transgenic plants expressing GFP fusion constructs to determine whether

bioinformatic separation of pollen tube reads from pistil reads accurately identifies

pollen tube-specific genes. We chose three MYB-dependent genes for which only the

pollen (Col) allele was detected in reads over SNPs and were thus deemed pollen tube-

specific (Figure 4A,H,O). We proposed that MYB expression, and thus target gene

activation, is induced by growth through pistil tissue (Leydon et al., 2013; Leydon et al.,

2014). To test this hypothesis and to determine whether RNA-seq analysis was

validated by reporter construct analysis, we analyzed GFP fusion proteins in the SIV

pollen tube growth assay and in wild-type pistils.

Full genomic loci containing the predicted promoter regions and coding

sequences of SUCROSE-PROTON SYMPORTER 9 (SUC9), PECTIN LYASE (PL), and

ALPHA CARBONIC ANHYDRASE 4 (ACA4) were cloned in frame with GFP to produce

translational fusions (Methods). We introduced these reporter constructs into

Arabidopsis (Col) carrying the pollen-specific LAT52:DsRed marker (Twell et al., 1989;

Francis et al., 2007), which drives expression of soluble DsRED in the pollen tube

cytoplasm. Mature pollen from each reporter was analyzed by confocal microscopy, but

GFP was not detectable, consistent with the hypothesis that these MYB-regulated

genes are induced by growth through pistil tissue. By contrast, after analyzing at least

21 independent primary transformants (T1) for each construct in SIV growth

19

experiments, we found that pollen-tube expression was observed in the vast majority of

transformants (SUC9:SUC9:GFP, 36/36; Pectate Lyase:GFP (PL:PL:GFP), 18/21;

ACA4:ACA4:GFP, 32/36; Figure 4B,I,P).

We determined the subcellular localization of each GFP fusion protein in growing

pollen tubes and each displayed a localization pattern consistent with the predicted

localization. SUC9:GFP appeared to localize predominantly to the plasma membrane,

and to vesicles in the pollen tube tip (Figure 4C, Supplemental Movie 1). PL:GFP was

observed in the endomembrane system (puncta) and what is likely the pollen tube cell

wall (Figure 4J, Supplemental Movie 2). ACA4:GFP was observed in the

endomembrane system (puncta, Figure 4Q, Supplemental Movie 3). In all SIV

experiments where GFP fluorescence was observed (86/93), expression was apparent

in extending pollen tubes and not pollen grains.

To determine whether these genes are pollen tube-specific, as they had been

designated by SNP-informed RNA-seq, we examined three independent transformants

for the expression of GFP unpollinated pistils using confocal microscopy. For each

transgenic line, we observed only autofluorescence in the stigma, style, transmitting

tract and all other somatic tissues of the pistil (Figure 4D,K,R). Surprisingly, we

observed expression of SUC9:GFP in the synergid cells (Figure 4D,E, Supplemental

Figure 7A–B,I) and ACA4:GFP in all cells of the female gametophyte (Figure 4R,S,

Supplemental Figure 7C–D,I); as expected, PL:GFP was undetectable in any pistil

tissue. We then analyzed self-pollinated pistils for each reporter construct (Figure

4F,M,T). Each reporter was most strongly detected in pollen tubes, especially in the

style region where pollen tubes are concentrated (arrows). Additionally, each reporter

20

fusion was detected in ovules clearly targeted by a pollen tube (Figure 4G,N,U),

supporting a possible role for these MYB-dependent genes in pollen tube reception

(Leydon et al., 2013).

Expression of SUC9:SUC9:GFP and ACA4:ACA4:GFP reporter constructs in the

female gametophyte was surprising in light of our SNP-based RNA-seq results, where

we detected no SUC9 or ACA4 Cvi SNP-containing transcripts. Transgenic lines were

generated in the Col accession, whereas our RNA-seq utilized Cvi as the pistil donor.

So, we tested whether expression of SUC9, PL, and ACA4 in the unpollinated pistil was

accession-specific using RT-qPCR. Of the three genes tested, only SUC9 transcripts

were detected at low levels in Col unpollinated pistils; none were detected in either Cvi

or Ler (ms1) unpollinated pistils (Supplemental Figure 8). This observation is consistent

with SUC9:SUC9:GFP expression being detected in synergid cells of transgenic Col

plants, but not in Cvi, the female used in our RNA-seq experiments. Interestingly, ACA4

transcripts were undetectable in unpollinated pistils of all three accessions, suggesting

that gametophytic expression of ACA4:ACA4:GFP may be due to inclusion or exclusion

of cis-regulatory regions in the promoter region of this transgene. Pectate lyase

transcripts were also undetectable in unpollinated pistils, consistent with our RNA-seq

results.

The observation that SUC9 was expressed in the synergid cells of Col but not

other accessions (Ler, Cvi), prompted us to test whether accession affected SUC9, PL,

and ACA4 expression in self- and cross-pollinations. Col self-pollinations result in higher

levels of each transcript compared with Cvi (Supplemental Figure 8). All three genes

behaved similarly in Col and Ws self-pollinations, except for SUC9, which was induced

21

at lower levels in Ws. Furthermore, Cvi x Col inter-accession pollinations induce SUC9,

PL, and ACA4 transcripts at lower levels than Col self-pollinations, suggesting that

pollen–pistil interactions, not pollen genotype alone, affect the strength of MYB-

dependent pollen tube transcriptional activation in vivo (Supplemental Figure 8). This

accession-specific activation of MYB-dependent genes led us to examine the role of

accession in reproductive gene expression in our RNA-sequencing data sets.

Gene expression changes are induced by inter-accession pollination.

Col and Cvi are inter-fertile; however, inter-accession crosses show subtle

defects in pollen tube reception that resemble pollen tube overgrowth defects in fer, lre,

or myb triple-mutant pollinations (Escobar-Restrepo et al., 2007; Capron et al., 2008;

Leydon et al., 2014; Muller et al., 2016). These observations along with the finding that

pollen tube-expressed genes are differentially expressed between accessions,

prompted us to determine whether there are genome-wide, accession-specific changes

in pollen and pistil gene expression. First, we used hierarchical clustering to assess

whether the transcriptomes of inter-accession pollinations were distinct from intra-

accession pollinations or unpollinated pistils. As expected, hierarchical clustering

demonstrates that pollinated pistils are distinct from unpollinated Cvi pistils (Figure 5A).

We compared the transcriptomes of self-pollinations (Cvi x Cvi, Col x Col) to inter-

accession pollinations (Cvi x Col), and found a core set of 16,838 genes expressed at

detectable levels in each pollination type (>100 reads, Figure 5B). However, differential

expression analysis identified hundreds of genes with significantly different levels of

22

expression between Col and Cvi self-pollinations (Figure 5C, Supplemental Data Set

10).

Accession-specific transcriptional responses have been previously observed

(Autran et al., 2011; Nodine and Bartel, 2012; Del Toro-De Leon et al., 2014), so it is not

surprising that Col and Cvi accessions display variation in transcriptional profiles of

reproductive cells and tissues. Interestingly, we identified members of gene families that

were enriched in the Col pollination transcriptome that were absent from Cvi pollinations

such as small secreted defensin-like proteins (DEFL, Figure 5D), low-molecular-weight

cysteine-rich proteins (LCR, Figure 5E), and NBS-LRR genes (TIR-NBS-LRR, Figure

5F). By contrast, analysis of genes enriched in Cvi pollinations, but absent in Col, did

not show enrichment for any particular GO-enrichment or gene families (see Methods).

However, despite the lack of enriched GO terms suggesting roles in extracellular

signaling, further analysis revealed that 44% of Cvi-specific pollination genes were

predicted to contain signal peptides, similar to ~31% found in Col-specific pollinations

(SignalP). These data suggest that Cvi may express its own repertoire of secreted

proteins during pollination, but that unlike Col, the gene families being expressed are

not as well annotated.

The pistil transcriptome responds to the pollen genotype during pollination. We

set out to determine whether the pistil transcriptome changes in response to pollen

accession in the pollinated pistil. Our analysis of myb triple-mutant pollinations

suggested the pistil responds differently to mutant versus wild-type pollen tubes (Figure

3C, E) and we reasoned the pistil may also mount unique responses to different pollen

23

accessions. We defined pollen tube and pistil changes in gene expression (DEGs) that

occur during intra-accession (Cvi x Cvi) versus inter-accession (Cvi x Col) pollinations

(Figure 5F, purple dots). First we ascertained genes that were differentially expressed

between self-crosses (Cvi x Cvi) and inter-accession crosses (Cvi x Col, Figure 5G,

Supplemental Data Set 10). This analysis identified 2,877 differentially expressed

genes; 1,238 are significantly more abundant in intra-ecotypic pollinations, 1,640 are

significantly higher in inter-ecotypic pollinations (632 up in inter-accession pollinations,

418 up in intra-accession pollinations). We then determined the parent of origin for

these genes and identified 1,050 pistil-expressed genes that are differentially expressed

depending on the pollen genotype (Figure 5H, red dots; Supplemental Data Set 10).

Most of the accession-sensitive transcriptional changes were subtle (< 4-fold). The 632

pistil genes that were differentially upregulated in inter-accession pollinations were

enriched for responses to various molecules (22.38 - enrichment score for this cluster of

responses), including chitin (n = 37, p – 2.6x10-25), carbohydrates (n = 39, p – 2.26x10-

20), and organic substances (n = 83, p – 1.2x10-15). Transcription factors were also

enriched (n = 61, p – 1.6x10-6). Interestingly, genes induced in the inter-accession (Cvi x

Col) pistil, included 14 ethylene-responsive transcription factors (ERFs) that are

implicated in pathogenesis-related transcriptional response (n = 14, p – 4.7x10-3,

Supplemental Data Set 11). The 418 pistil-expressed genes that were enriched in intra-

accession crosses (Cvi x Cvi) were not strongly enriched for these or other functional

categories. This subtle difference in transcriptional profiles indicates that pollen

accession is perceived by the pistil, activating genes associated with defense responses

during inter-accession pollinations.

24

Expression of a large family of thionin-like CRPs is MYB-dependent and

accession-specific.

Previous analysis of myb triple-mutant pollinations using microarray analysis had

identified a single thionin-like gene (AT5G36720) as being among the most differentially

regulated between mutant and wild-type pollinations (Leydon et al., 2013). This

prompted us to propose that these proteins may be secreted by pollen tubes to female

gametophytes to mediate pollen tube reception signaling. However, analysis of this

family (cysteine rich protein 2460, CRP2460, Silverstein et al., 2007) indicated that

many members of this large group of genes were not annotated and therefore were not

represented on the ATH1 array. We analyzed this family using our RNA-seq data to

determine whether multiple members were potentially MYB-regulated and to determine

whether their expression varied among accessions. We plotted the RNA expression of

each family member alongside the CRP2460 phylogenetic tree (Figure 6, Supplemental

Data Set 12). Differential expression analysis identifies 22/28 CRP2460 transcript levels

as statistically lower in pollinations with myb triple-mutant pollen tubes (Figure 6B,

asterisk) compared to wild type. CRP2460 thionin proteins were expressed accession-

preferentially: high expression was observed in Col pollen tubes, while Cvi pollen tubes

expressed fewer members and at lower levels (Figure 6B). Of the 28 thionin-like

proteins analyzed, 15 contained polymorphisms that allowed us to determine whether

expression of these genes was from the pollen tube or pistil (Figure 6B, +). In each

case, all reads over SNPs carried the Col allele, indicating that thionins are pollen tube

expressed (Figure 6C). Thionin proteins therefore represent a pollen tube expression

25

signature that differentiates Arabidopsis accessions from each other and may be

involved in signaling between the pollen tube and the female gametophyte important for

sperm release.

MYB-dependent, pollen tube-expressed genes show elevated polymorphism

rates, gene family expansion, and positive selection. myb triple-mutant pollen tubes

fail to release sperm and continue to grow within the female gametophyte (Leydon et

al., 2013; Liang et al., 2013). This defect, first identified in the Arabidopsis feronia

mutant (Huck et al., 2003; Rotman et al., 2003), had been characterized as a pre-

zygotic barrier to hybridization in interspecific cross-pollinations (Williams et al., 1986;

Escobar-Restrepo et al., 2007; Muller et al., 2016) and suggests that MYB-dependent

genes may be involved in shaping the species identity of the pollen tube that is

recognized by the female gametophyte (Leydon et al., 2014). Molecules involved in

gamete:gamete recognition have been shown to evolve rapidly and to be responsible

for species-level discrimination in mating interactions (Metz et al., 1998; Clark et al.,

2006). Interestingly, it was recently shown that the pollen tube expresses a larger

fraction of species-limited genes than other tissues (Arunkumar et al., 2013; Gossmann

et al., 2014), which is consistent with the idea that pollen tubes may express species-

limited proteins that determine their species-level identity. To address whether groups of

genes expressed in reproductive tissues maintain higher rates of sequence diversity, we

calculated Col v. Cvi exonic SNP rates and compared them to control rates simulated

using 100 sets of randomly selected genes of the same number. We used the ratio of

26

the measured SNP-rate to the simulated rate to identify groups of genes with elevated

SNP rates (Table 1, Supplemental Data Set 13).

Analysis of groups of genes defined by their expression pattern during pollination

without determining parent of origin (analysis of all reads) revealed that the ~10,000

genes expressed in the pollinated pistil have SNP rates that are significantly below the

simulated rate. However, genes induced by pollination contain significantly higher than

control rates (Table 1). The 292 genes that are differentially expressed in myb triple-

mutant pollinations (identified from all mapped reads) do not show elevated SNP rates.

When analyzed in bulk, we only observe increased polymorphism rates in genes

induced by pollination.

However, when sex-specific reads are analyzed to focus on changes occurring in

pollen and/or pistil gene expression, three groups were found to have polymorphism

rates elevated compared to the simulated rate: pollen expressed genes, pistil-specific

pollination induced genes, and MYB-regulated genes (Table 1). To gain insight into

MYB-regulated genes with elevated polymorphism rates, we focused on a MYB-

regulated CRP gene family (Figure 6). We started by analyzing the NBS-LRR gene

family, which was previously found to have high rates of major effect SNPs in

Arabidopsis accessions indicative of diversifying selection (Clark et al., 2007; Schmitz et

al., 2013) and found a ratio of measured SNP rate to control rate (1.24, Table 1) that

was significantly above one. A significantly high SNP rate (p<0.01, Poisson distribution)

was also observed for a group of 825 CRP encoding genes, but not for the thionin

subgroup (n = 71). We also determined the frequency that SNPs in these groups of

genes resulted in missense mutations. Interestingly, the thionin subclade of CRPs had

27

the highest rate of missense mutations (Table 1, Supplemental Data Set 14),

suggesting this group of genes may be under diversifying selective pressure.

To test for positive selection within thionins (CRP 2200-2610), we first

determined whether there were homologs for each gene in Arabidopsis lyrata, Populus

trichocarpa, Vitis vinifera, Oryza sativa, and Physcomitrella patens using a recently

published genome-wide analysis (Supplemental Data Set 15, Lloyd et al., 2015).

Appreciable fractions of Actin and NBS-LRR gene families have homologs in distantly

related species such as the monocot, O. sativa, and the moss, P. Patens (Figure 7A).

By contrast, homologs were only identified in closely related A. lyrata for the vast

majority of CRP2200-2610 genes (Figure 7A). For example, 30% of NBS-LRR genes

have homologs in P. patens, whereas no homologs for CRP2200-2610 genes were

identified (Lloyd et al., 2015). Moreover, only a small subset of the CRP2200-2610

genes have homologs in angiosperm species outside the Brassicaceae (P. trichocarpa,

V. vinifera, and O. sativa), suggesting a recent expansion of this group.

To profile diversification in these families, we plotted the mean Ka/Ks for genes

with homologs; we found that Actin genes had lower rates than NBS-LRRs, consistent

with the hypothesis that NBS-LRR genes are under diversifying selection, while the

Actin genes are under purifying selection (Figure 7B). CRPs exhibit a Ka/Ks profile that

is similar to NBS-LRR genes (Figure 7B). However, this analysis of mean Ka/Ks failed

to highlight the elevated proportion of CRP gene family members (CRPs in general and

CRP2200-2610 in particular) with a Ka/Ks above one in the comparison with closely

related A. lyrata, which had the largest number of homologs (Figure 7C).

28

This analysis revealed a dramatic expansion of the CRP2200-2610 thionin clade

of CRPs in the Brassica lineage. Of particular interest is the CRP2460 (Figure 6) group,

which has undergone an expansion in A. thaliana compared with the closely related A.

lyrata. Only six of 28 genes within this group have homologs in A. lyrata, and none have

homologs in any of the other species tested (Figure 6, Ka/Ks indicated). Furthermore,

the Ka/Ks rate for At1g21835 compared to its homolog from A. lyrata was found to be

above one (Ka/Ks = 1.06, Figure 6), suggesting selective pressure to maintain diversity

of this gene. At1g21835 appears to be the founder of an expanded group of Arabidopsis

thionins, because it is sister to 14 genes without homologs in A. lyrata (Figure 6,

Supplemental Figure 9). Analysis of this expanded group revealed evidence for

diversifying selection at specific sites within these ~100-amino acid proteins

(Supplemental Figure 9, Supplemental Data Set 15). These results are in keeping with

our proposal that this group of secreted proteins convey species-unique information to

the female gametophyte required for pollen tube reception.

Discussion

Successful plant reproduction is the lynchpin of agricultural food production, so the

molecular mechanisms responsible for pollen–pistil signaling pathways need to be

established so they can be optimized for grain and seed production in a changing

climate. Moreover, it is clear that pollen tube–pistil interactions represent a key pre-

zygotic barrier to interspecific hybridization; however, few molecules have been

identified that facilitate sperm delivery in intraspecific pollinations, while preventing

sperm delivery in interspecific pollinations (Moyle et al., 2014). Determination of the

29

pollen tube and pistil transcriptomes during pollination would facilitate identification of

patterns of gene expression required for successful fertilization; however, this has been

technically challenging because of the way that pollen tubes grow within complex pistil

tissue. Here, we present a method to circumvent this challenge that takes advantage of

SNPs to define pollen tube- and pistil-derived RNA-seq reads within the pollinated pistil.

This method does not require transgenic plants or invasive procedures and is therefore

translatable to crop species with sufficient diversity among cross-compatible

accessions/cultivars. We validated this method using comparisons to previous data sets

and by testing the expression patterns of pollen tube-specific genes using transgenic

reporter constructs. We have successfully defined the relative contributions of the pollen

tube and pistil to the pollination transcriptome and reveal pistil-expressed genes that

respond specifically to the genotype of the pollen tube. Moreover, we defined patterns

of pollen tube gene expression controlled by MYB97, MYB101, and MYB120, pistil-

induced pollen tube transcription factors required for successful sperm release. This

analysis revealed that pollen tube MYB transcription factors are required for the

expression of secreted proteins, including small cysteine-rich proteins that may play

critical roles in signaling to female cells to initiate sperm release. Finally, we present

evidence that patterns of pollen tube and pistil gene expression are variable between

Arabidopsis accessions and that pollen tube-expressed genes are more highly

polymorphic than other groups of genes. These data provide a framework for further

analysis of the patterns of gene expression that promote successful sperm delivery in

intraspecific pollinations while preventing sperm delivery in interspecific crosses.

30

The modular nature of pollen and pistil, which facilitates pollination between

polymorphic parents, combined with the development of RNA-seq techniques provided

a means to comprehensively define pollen and pistil transcriptomes during pollination.

Arabidopsis accessions are of great utility for this approach because pollen and pistil

can be easily crossed in any combination. We chose Cvi and Col as the initial parental

accessions because of their relatively high polymorphism rate (~1 SNP/200 nucleotides)

and found this combination was sufficient to track patterns of pollen- and pistil-specific

gene expression for ~60% of the genes expressed in the pollinated pistil (Figure 1F).

This leaves a significant fraction of genes whose parent of origin cannot be defined in

this particular cross (see Supplemental Figure 4 for analysis of accession combinations

providing additional coverage). Despite this limitation, the ease of experimental setup

and non-invasive nature of the technique argue for this approach, which does not

require the extensive post-harvesting purification steps required for alternative

biochemical approaches such as TRAP-seq (Lin et al., 2014; Kim et al., 2015) or

isolation of nuclei tagged in specific cell types (INTACT, Deal and Henikoff, 2011). Our

analysis of Cvi x Col cross-pollinations revealed that ~8% of pollinated Arabidopsis pistil

RNA is derived from the pollen tube and we found strong correlations between our

analysis and previous microarray analyses of the component cell types (microarray

experiments recently reviewed in (Rutley and Twell, 2015, Figure 2). Importantly, we

identified hundreds of novel pollen tube- and pistil-expressed genes and showed that

SNP-informed RNA-seq analysis of pollination can be used to identify promoter

elements that drive gene expression in the pollen tube in response to growth through

the pistil (Figure 4).

31

It is becoming clear that the near simultaneous rupture of the pollen tube and

receptive synergid cell (Leydon et al., 2015) are the consequence of highly coordinated

cell–cell signaling requiring pollen tube and synergid transcriptional networks (Leydon et

al., 2013; Liang et al., 2013; Mendes et al., 2016), expression of cell surface signaling

molecules (Escobar-Restrepo et al., 2007; Kessler et al., 2010; Lindner et al., 2015; Liu

et al., 2016), and Ca2+ fluxes that define which of two synergids receives the pollen tube

(Denninger et al., 2014; Ngo et al., 2014). Using SNP-informed RNA-seq analysis of

myb triple-mutant compared with wild-type pollinations, we have provided a

comprehensive set of genes expressed by pollen tubes that mediate interactions with

female cells required for pollen tube burst, sperm release and synergid degeneration.

Our analysis defined three main categories of genes regulated by MYB97, MYB101 and

MYB120: transmembrane transporters, carbohydrate active proteins and secreted

proteins of diverse function. This result was consistent with the enriched GO categories

previously identified by microarray analysis (Leydon et al., 2013). However, RNA-seq

analysis revealed many novel MYB-dependent genes in these categories that are

members of large gene families and were not detectable by microarray analysis

because hybridization-based probes could not differentiate closely related family

members or because the genes were not annotated at the time of microarray

production. The thionin-like family of small cysteine-rich proteins provides a striking

example: a single MYB-dependent member was identified by microarray analysis

(Leydon et al., 2013) while RNA-seq analysis identified 24 family members whose

expression was significantly decreased in myb triple-mutant pollinations compared with

wild type (Figure 6).

32

MYB-dependent secreted proteins were of particular interest because recent

analysis documented the rich diversity of the tobacco pollen tube secretome (Hafidh et

al., 2016) and suggested that the pollen tube and the synergid cell (Punwani et al.,

2007; Liu et al., 2015) both evolved highly active secretory systems. Interestingly, genes

that require MYB transcription factors for expression in the pollen tube are enriched for

secreted proteins including two alpha carbonic anhydrases (Figure 4O-T), 11

endopeptidases, 22/28 thionins in the CRP2460 gene family (Figure 6), 19/98 S-protein

homologs (Ride et al., 1999), two cysteine-rich secretory, antigen 5, and pathogenesis-

related 1 proteins (CAP; AT3G09590, AT5G02730), and four rapid alkalinization factors

(RALFs; AT2G32835, AT2G32788, AT2G32785, AT1G60815, Supplemental Data Set

8, 9). Interestingly, a comparison between the tobacco pollen tube and Arabidopsis

MYB-dependent pollen tube genes revealed significant overlap both in terms of specific

gene homologs and for functional gene categories (Hafidh et al., 2016). These findings

suggest a core group of proteins secreted by pollen tubes of dicot species that could be

important for sperm release.

The pollen transcriptome is overrepresented by carbohydrate active enzymes

involved in cell wall synthesis (Honys and Twell, 2004; Pina et al., 2005; Qin et al.,

2009), an enzymatic function that was enriched in pollen tube-expressed genes

identified in our RNA-seq experiments (Supplemental Data Set 6). Our analysis also

found that the MYB-dependent transcriptome is enriched for secreted carbohydrate

active proteins such as pectate lyase (n = 6, Figure 4, Supplemental Data Set 8, 9) and

pectin methylesterase inhibitors (n = 4, Supplemental Data Set 8, 9), both of which are

predicted to modify pectin, a major component of the pollen tube wall. Regulation of

33

pectin structure at the pollen tube tip has been shown to be essential for pollen tube

growth; mutants lacking pectin methylesterases (enzymes facilitating pectin cross-

linking), rupture in vitro and in the pistil (Bosch et al., 2005; Jiang et al., 2005). Unlike

mutants defective in pectin methylesterases, myb triple-mutant pollen tubes do not have

growth defects in vitro and do not rupture prematurely in the pistil; instead, they fail to

burst upon contact with the synergid (Leydon et al., 2013; Liang et al., 2013). This

phenotype suggests that carbohydrate active enzymes regulated by MYB transcription

factors could be required for regulated cell wall instability upon contact with synergids,

or they could determine a cell wall carbohydrate profile that is recognized by the

synergid to facilitate sperm release by pollen tubes of the same species.

When we compared the Cvi and Col self-pollination transcriptomes, we found

evidence for a core set of pollen tube-expressed genes, but also many examples of

quantitative and qualitative differences in reproductive gene expression (Figure 5).

These data are consistent with the idea that patterns of reproductive gene expression

diversify rapidly when populations are geographically isolated. Defining the functions of

accession-specific genes we identified here will inform efforts to understand the

mechanisms of pollen tube growth, guidance and reception, which appear to evolve

rapidly and create barriers to interspecific hybridization (Escobar-Restrepo et al., 2007;

Takeuchi and Higashiyama, 2012). Interestingly, we observed families of MYB-

regulated, small secreted proteins that were overrepresented in Col self-pollinations, yet

absent from Cvi pollinations. This suggests that mechanisms for pollen tube reception

and sperm release may be tuned to a particular accession and are in keeping with our

prior observation of pollen tube reception defects in inter-accession crosses (Leydon et

34

al., 2014). Concordantly, we also observed many genes that were expressed in Cvi

pollinations that were absent in Col pollinations and lacked any usable GO-terminology.

Additional analysis of these genes showed that many contain predicted signal peptides,

and may be members of as yet undefined gene families.

Comparison of self-pollinations with inter-accession pollinations revealed a small

set of Cvi pistil-expressed genes that are induced by Col pollen tube growth, but not by

Cvi pollen tube growth. These genes were enriched for functions associated with

defense responses, especially an ethylene-responsive transcriptional response

(Supplemental Data Set 11). Therefore, we propose that accession-specific expression

of a complement of secreted proteins controls the interaction of the pollen tube with

pistil cells and directs the pistil’s response between reproduction and defense

depending on the pollen tube genotype.

Reproductive genes have been shown to be rapidly evolving in many species

(Clark et al., 2006). Some of the best examples include sperm proteins with high rates

of nonsynonymous substitutions in aquatic species with motile sperm that spawn at the

same time as closely related sympatric species (Metz et al., 1998). It has been

proposed that rapid co-evolution between sperm surface proteins and their egg-

expressed binding partners provides a barrier to interspecific gamete fusion. In

flowering plants, the pollen tube is motile and performs the required cell–cell

interactions with female tissues (the pistil) that determine male reproductive recognition

and fitness – not the sperm as in animals (Clark et al., 2006). Indeed, we find that SNPs

are enriched 1.5x over controls in pollen-expressed genes (Col to Cvi), and these SNPs

are 1.7-1.9x more likely to cause a missense mutation (Table 1). Strikingly, the MYB-

35

regulated pollen tube-specific genes that contain SNPs carry 1.7x more SNPs than the

background rate, providing sequence diversity on which evolution can act to select for

pre-zygotic barriers to inter-specific reproduction. Pistil-specific genes that are

pollination responsive also display increased SNP-rate and missense frequencies

(Table 1).

We analyzed a group of small secreted, MYB-dependent CRPs (CRP2460) to

determine whether their patterns of evolution are consistent with a role in determination

of species-level identity of the pollen tube during interactions with the synergid cell. A

MYB-dependent member of this group (At5g36720), was previously shown to be

expressed in the pollen tube and localized to the pollen tube secretory system (Leydon

et al., 2013). This group is expanded in A. thaliana relative to A. lyrata and homologs

were not found outside of the Brassicaceae (Figure 6, Figure 7, Supplemental Figure 9).

Moreover, a comparison of the At1g21835 gene and its A. lyrata homolog revealed a

Ka/Ks value greater than one, suggesting selective pressure to maintain amino acid

diversity. Intriguingly, this gene is sister to a clade of 14 A. thaliana genes with no

homologs (Supplemental Figure 9A) in A. lyrata, consistent with a very recent A.

thaliana expansion. Analysis of individual residues revealed evidence for diversifying

selection interspersed with purifying selection to maintain the position of cysteines likely

to be important for maintaining structural integrity of the proteins (Supplemental Figure

9B, Supplemental Data Set 15). Future experiments will be directed toward determining

whether this group of secreted proteins is a critical component of the MYB-controlled

pollen tube–pistil recognition system required for sperm release.

36

SNP-informed RNA-seq analysis of pollination provides a powerful method to

quantify pollen–pistil interactions over any time-course to define sex-specific

transcriptional responses. We have used this method to define the set of genes

controlled by pollen tube MYB transcription factors MYB97, MYB101, and MYB120 that

mediate signaling between the pollen tube and synergid cell required for sperm release.

However, this method could be used to analyze any pollen tube or pistil mutant affecting

pollen–pistil interactions. Moreover, this method lends itself readily to analysis of the

impact of environmental change on the reproductive transcriptome of crop plants and

holds the potential to uncover how pollen tube–pistil interactions change in response to

abiotic and biotic stresses that limit crop productivity.

37

Materials and Methods Plant Material

Seeds were grown on 0.7% agar Murashige and Skoog (MS) medium (Sigma Aldrich,

St. Louis, MO). Arabidopsis thaliana plants were grown in chambers at 21°C under

illumination (100 µmol m-2 s-1 with a 16-h photoperiod). Seedlings were transplanted to

sterile 2MIX potting media (www.fafard.com) between 7 and 10 days after plating. All

plants were grown at 20°C, 50-60% humidity, on a 16h / 8h day/night cycle in growth

chambers (Environmental growth chambers, Chagrin Falls, OH, USA). SIV pollinations

were performed as previously described (Palanivelu and Preuss, 2006; Qin et al.,

2009).

mRNA-seq library construction

RNA was isolated from excised pistil tissues either unpollinated or 8 h after pollination

using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs,

Ipswich, MA). At least 10 µg of total RNA was oligo-dT-bead purified and RNA-

sequencing libraries were prepared according to the NEBNext protocol. Primers 4, 6,

and 12 were used from NEBNext® Multiplex Oligos for Illumina (New England Biolabs,

Ipswich, MA). Amplification was for 15 cycles. Libraries were tested by qPCR after

amplification to validate amplification.

mRNA-seq data analysis

Pistils were collected with and without pollination in triplicate (15 samples). Single-end

sequencing was performed with mRNA-seq libraries on an Illumina HiSeq machine.

38

Read length was 50 bp. Sequencing quality was analyzed using fastqc. Reads were

aligned to the Arabidopsis genome (TAIR10) using Tophat v2.0.13 (Kim et al., 2013).

Tophat options were --segment-length 22 --segment-mismatches 1 --max-segment-

intron 11000. Reducing segment length or mismatch number yielded no additional

mapped reads, indicating that this segment length, mismatch number was optimal for

mapping our libraries to the TAIR10 genome. RNA-seq data have been deposited at the

Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/ ,NCBI BioProject:

PRJNA329634; SRA Accession: SRP078926). Aligned reads were parsed using a

PERL script released by the Ghering Lab (Pignatta et al., 2014). Read counts per gene

were quantified using HTSeq (Anders et al., 2015) and EasyRNASeq (Delhomme et al.,

2012). Multiply-mapping reads were counted because many pollen expressed genes

were observed to be in redundant gene families due to gene duplication.

GO analysis

GO analysis was performed using the DAVID bioinformatics resource version 6.7

(Huang et al., 2009). Reported p-values were corrected using the Benjamini method.

Calling variants

RNA sequencing reads from unpollinated Cvi-0 and Cvi-0 8 h after self-pollination was

analyzed for novel SNP variants following the GATK Best Practices workflow (McKenna

et al., 2010). Briefly, mapped reads (Tophat2) were prepared for variant calling: Read

group addition, mark duplicates, Split’N’Trim, ReassignMappingQuality, Indel

Realignment, Base Recalibration. Variant calling was performed using the GATK

HaplotypeCaller. Variant filtration was performed (FS>30, QD<2.0). SNPs supported

39

with less than 10 reads or that were not homozygous were discarded. Any SNPs in

common with previously established Cvi-0 SNPs (Pignatta et al., 2014) were considered

validated and set aside from the list of novel SNPs. Unpollinated Cvi-0 and Cvi-0 self-

pollinated RNAseq library reads were then run against this novel SNP list using a

previously published Perl pipeline (Pignatta et al., 2014). Any SNP locations that

returned a Col-0 SNP were discarded from the BED file. This eliminated 2000 SNPs,

and created a final additional 15995 SNPs (in 237 genes previously without

documented SNPs, and 5217 genes that had one or more previously described SNP).

Variant analysis

The effect of SNPs was predicted using SnpEff and SnpSift software (Cingolani et al.,

2012a; Cingolani et al., 2012b). Master SNP lists were correlated to the TAIR10 gene

annotation file to identify the distribution of SNPs in genomic elements using a range

overlap function via the IRanges package (Lawrence et al., 2013) and a custom R script

(R Core Team (2013), R: A language and environment for statistical computing; R

Foundation for Statistical Computing, Vienna, Austria. (http://www.R-project.org/). The

SNP rate was computed for each gene (Rate = SNPs/nucleotide), and the average SNP

rate of individual gene lists was compared to background frequencies simulated using

randomly generated lists of the same size as input lists. Each comparison background

frequency was the average of 100 simulations. Similarly, the rate of missense mutations

was calculated for each gene and averaged for the individual gene lists. These

frequencies were compared to the rate of missense mutations across the entire

40

genome. R scripts and data for analysis are part of the package SexSpecificExpression,

which is available on GitHub at https://github.com/evenable/SexSpecificExpression.git.

Transcriptional profile correlation analysis

Pairwise correlations were generated between our RNA-seq data and microarray

data from previous studies: Data on uninucleate microspores, tricellular pollen, and

mature pollen grains are from Honys and Twell (2004). Data on dry pollen, 0.5 h, 4 h

and SIV-grown pollen tubes are from Qin et al. (2009). Data from unpollinated &

pollinated pistils, and ovules are from Boavida et al. (2011). Data from stigma and ovary

are from Swanson et al. (2005). Data from roots, seedlings, and rosette leaves are from

Schmid et al. (2005). For each pair, Pearson-r was computed over all genes with a

unique SNP, using mean reads from three biological replicates for RNA-seq and mean

absolute intensity for multiple microarray replicates.

Graphing sequencing data

Scatterplots were generated from sequence data using Python’s Matplotlib module.

Analysis of differential gene expression

Three differential gene expression callers, DESeq, DESeq2, and EdgeR (Anders and

Huber, 2010; Robinson et al., 2010; Love et al., 2014), were tested to define

differentially expressed genes using the Cvi x Col vs Cvi x myb triple-mutant as a test.

DESeq differential expression was performed using the default parameters as per the

DESeq vignette (estimateSizeFactors, estimateDispersions, nbinomTest, Anders and

Huber, 2010). Differential expression was performed using DESeq2 and a trimmed

41

mean approach (replaceOutliersWithTrimmedMean, Love et al., 2014). Data were

visualized according to the vignette for DESeq2. Differential expression with EdgeR was

performed as per the EdgeR vignette with the user defined estimateGLMCommonDisp -

Estimate Common Dispersion for Negative Binomial GLMs, estimateGLMTrendedDisp -

Estimate Trended Dispersion for Negative Binomial GLMs, estimateGLMTagwiseDisp -

Empirical Bayes Tagwise Dispersions for Negative Binomial GLMs functions. All

subsequent differential expression analysis was performed using DESeq2 as described

above.

GFP fusion constructs

To generate ACA:GFP and PL:GFP constructs, genomic fragments containing the

promoter (0.96 kb upstream of the ACA start codon; 1.18 kb upstream of the PL start

codon), introns and exons (excluding the stop codon) were amplified by polymerase

chain reaction and inserted into the pENTR-D vector (Invitrogen). Genomic clones in

pENTR-D were integrated into pMDC107 using LR Clonase II (Invitrogen). Genomic

SUC9:GFP was generated by recombining a genomic clone in pCR8/TOPO/GW (Sivitz

et al., 2007) with pMDC107. Genomic clones were electroporated into Agrobacterium

strain GV3101, and transformed into the LAT52:dsRed, qrt1-2 reporter line by floral dip

{Clough, 1998 #1302}. Primary transformants were selected by hygromycin resistance

and analyzed for expression by SIV-PT growth experiments. To generate the nuclear-

localized GFP reporter under the MYB98 promoter, the promoter sequence {Kasahara,

2005 #1282} was amplified from wild-type Col-0 genomic DNA and inserted into the

Greengate pGGA000 entry vector (Greengate citation). Using a Golden Gate Assembly

42

method, the promoter was fused to a nuclear-localized GFP (nGFP), terminator

sequence, and Glufosinate ammonium (Basta) herbicide resistance cassette

(pGGB003, pGGC012, pGGD002, pGGE009, pGGF001) and cloned into the

destination vector (pGGZ003). Primary transformants were isolated through Basta

resistance, and screened for nGFP expression in female gametophytes. All

oligonucleotides used are given in Supplemental Table 1.

RNA extraction and real-time quantitative PCR

RNA was purified using the Qiagen RNeasy Kit (http://www.qiagen. com). The yield and

RNA purity were determined by Nano-Drop (Thermo Scientific, Wilmington, DE, USA).

For reverse transcription quantitative PCR (RT-qPCR), RNA was treated with DNAse

using the Ambion TURBO DNA-free Kit from Invitrogen (AM1907). Primers were

designed at Primer3Plus (primer3plus.com/), using qPCR-optimized settings. All

primers were tested to assure that they have near 100% efficiency and linearity prior to

use with an ABI 7000 real-time PCR machine. Three technical replicates for each of

three biological replicates were performed for each RT-qPCR experiment. The

comparative Ct method (User Bulletin #2, Applied Biosystems) was used for

quantification. Expression was normalized to the ubiquitous PP2AA3 gene. Real-time

quantitative PCR was performed with cDNA libraries using primers of equivalent

efficiency in triplicate reactions using SYBR Green qPCR Master Mix (Invitrogen) on an

ABI 7000 PRISM Sequence Detection System (Applied Biosystems). All

oligonucleotides used are given in Supplemental Table 1.

43

Imaging of pollen tube growth

For live imaging, emasculations, dissections and pollinations were performed under a

dissecting microscope (Zeiss Stemi 2000C). Ovary walls were removed 12 h post-

pollination as previously described (Leydon et al., 2013). Dissected pistil tissues were

mounted in 80 mM sorbitol for analysis by confocal laser scanning microscopy using a

40x objective with DIC capability (Zeiss LSM510 Meta inverted microscope). Standard

emission and detection wavelength settings were used to image GFP expression (argon

laser 458/477/488/514 nm at 30 mV) and dsRed/mRFP expression (helium neon laser

633 nm at 3 mV). Signal intensities were optimized for each individual fluorophore and

then combined in overlay. Final images represent a merge of single planes at varying

depths (z-stacks). Semi in vivo pollen tube imaging was performed on a Zeiss Lumar

V12 Fluorescence Stereomicroscope.

Phylogenetic Analysis

Sequence alignments were performed by ClustalW alignment in MEGA6 (Tamura et al.,

2013). Diversifying selection was tested for CRPs using the trees constructed using a

Maximum likelihood generated tree, estimated selection at codons (HyPhy), and

Estimated position-by-position mean evolutionary rate (Maximum likelihood) in MEGA6.

44

Accession Numbers

Gene sequence data from this article can be found in the GenBank/EMBL databases

under the following accession numbers: SUC9, AT5G06170; PL, AT5G09280; ACA4,

At4G20990. Other gene accession numbers are provided in Supplemental Data Sets 3–15.

RNA-seq data have been deposited at the NCBI Sequence Read Archive, BioProject:

PRJNA329634; SRA Accession: SRP078926.

Supplemental Materials

Supplemental Figure 1. SNP frequency in genomic features. Supplemental Figure 2. Flowchart of data analysis utilized to discover sex-specific differential gene expression. Supplemental Figure 3. Quantitative analysis of reads with SNPs suggests they are distributed across transcribed regions of genes similarly to reads without SNPs. Supplemental Figure 4. Predicting optimal accessions to detect pollen tube expressed transcripts. Supplemental Figure 5. Sex-specific gene expression profiles correspond to known reproductive transcriptional profiles. Supplemental Figure 6. Comparison of differential expression callers’ ability to identify MYB-dependent genes. Supplemental Figure 7. Expression of SUC9:GFP and ACA4:GFP in female gametophytic cells. Supplemental Figure 8. Accession-specific gene expression of MYB-dependent genes. Supplemental Figure 9. Rapid evolution of the CRP2460 thionin-related proteins by expansion and positive selection. Supplemental Table 1. Oligonucleotides used in this study. Supplemental Data Set 1. Final SNP database.

Supplemental Data Set 2. Table of mRNA-seq libraries generated in this study.

Supplemental Data Set 3. Table of Read counts per gene for Cvi x Col inter-ecotype

pollinations.

Supplemental Data Set 4. Genes differentially expressed during pollen tube growth in

the pistil.

45

Supplemental Data Set 5. Intersection and Venn Diagram of SIV-expressed genes.

(microarray) and pollen tube expressed genes from our SNP-Seq study.

Supplemental Data Set 6. Pollen-specific Genes: Gene Ontology.

Supplemental Data Set 7. Pistil-specific genes that are differentially expressed after

pollination.

Supplemental Data Set 8. Detecting differential expression of MYB-dependent genes. Supplemental Data Set 9. Quantification and classification of MYB-dependent sex-

specific genes.

Supplemental Data Set 10. Genes differentially expressed between Col x Col and Cvi

x Cvi pollinations.

Supplemental Data Set 11. Pistil expressed genes with elevated transcript levels in

inter-ecotype pollinations.

Supplemental Data Set 12. Alignment of thionin CRPs used to produce the

phylogenetic tree (Figure 6).

Supplemental Data Set 13. Gene lists used in Table 1: SNP rates are elevated in

MYB-dependent pollen-specific genes.

Supplemental Data Set 14. Annotation of Cvi SNPs to Col with strong effects on coding sequences and gene function. Supplemental Data Set 15. Analysis of natural selection in MYB-dependent CRP2460 family. Supplemental Movie 1. Live imaging of SUC9:SUC9:GFP SIV-grown pollen tubes.

Supplemental Movie 2. Live imaging of PL:PL:GFP SIV-grown pollen tubes.

Supplemental Movie 3. Live imaging of ACA4:ACA4:GFP SIV-grown pollen tubes.

Supplemental Movie Legends. Acknowledgements

We thank Mary Gehring (Whitehead Institute, MIT) for generous access to bioinformatic

resources and Christoph Schorl (Brown University Genomics Core Facility) for RNA-

sequencing. National Science Foundation Grant IOS-1353798 funded this project.

46

Author Contributions ARL performed the experiments. EV and CW performed the bioinformatics analyses.

AR and JW provided input and reagents for the SUC9 experiments. MJ and ARL

conceived the study, designed the experiments, and wrote the manuscript.

47

Table 1. SNP rates are elevated in MYB-dependent pollen-specific genes.

Single Nucleotide Polymorphisms (SNPs)

Missense mutations

# of

genes in group

Simulated Rate

Actual Rate

Ratio AR/SR

Actual Rate

Ratio AR/BR

All Reads

Pollinated Pistil Expressed 10,029 4.4x10-03 3.1x10-03 0.70* 1.99x10-03 2.19

MYB-Regulated 292 4.3x10-03 4.2x10-03 0.97* 7.29x10-04 0.80

Pollination Induced 1,058 4.3x10-03 6.2x10-03 1.44* 1.96x10-03 2.15

Sex-specific Reads

Pistil Expressed (Cvi Reads) 15,915 4.3x10-03 4.5x10-03 1.03* 1.83x10-03 2.01

Pollen Expressed (Col Reads) 4,375 4.3x10-03 6.6x10-03 1.53* 1.65x10-03 1.81

Pistil-Specific Pollination Induced (Cvi Reads) 254 4.4x10-03 7.0x10-03 1.59* 1.27x10-03 1.39

MYB-Regulated (Col Reads) 268 4.4x10-03 7.4x10-03 1.71* 1.16x10-03 1.28

Randomly Sampled SNP-Containing Genes 268 (x10) 4.4x10-03 4.5x10-03 1.06* 1.27x10-03 1.40

Selected Gene Families

NBS-LRR 167 4.2x10-03 5.3x10-03 1.24* 1.44x10-04 0.16

CRPs 825 4.3x10-03 5.1x10-03 1.17* 1.48x10-03 1.63

CRP2200-2610 (Thionin) 71 4.4x10-03 4.9x10-03 1.11 7.22x10-03 7.94

Gene Lists - See Supplemental File S12. Rate = SNPs/nucleotide, Ratio = see color scale. Simulated lists (100 randomly chosen gene lists of equal gene number to input set) were used to create a ratio of list rates to demonstrate SNP enrichments, see color scale. AR/SR – Actual rate/simulated rate. Ratios that have an asterisk (*) are statistically significant, p<0.01, Poisson distribution. Missense mutation rate = Missense mutation/nucleotide. Background rate for Missense mutations = 9.1 x10-04. AR/BR – Actual rate/Background rate.

48

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Figure 1. Pollen- and pistil-specific gene expression can be distinguished in pollinated pistils using SNPs in RNA-seq reads. A. Experimental samples prepared for RNA-seq. B. The distribution of SNPs across indicated gene regions in our Cvi v. Col SNP database (% is given above bars, black represents transcribed regions, gray represents untranscribed regions of transposable elements, TE). C. Reads from three biological replicates of a pollinated pistil RNA-seq experiment were analyzed to determine the number with Col or Cvi SNPs; the numbers of total and sex-specific reads are given. Venn diagram is not proportional to number of included elements. D and E. Scatterplots of mean reads/gene (3 biological replicates). D. Pistil reads (y-axis) are plotted against pollen reads (x-axis) for all genes with SNPs detected in pollinated pistils. The y-axis is 10-fold greater than the x-axis. E. Total reads/gene are plotted for unpollinated pistils (y-axis) and pollinated pistils (x-axis). All genes are plotted as colored dots indicating sex-specific expression: Blue, pollen specific; Red, pistil specific, Gray, genes without SNPs. F. Quantification of Genes expressed in pollen, pistil or in both tissues. Supporting bioinformatic analysis is shown in Supplemental Figures 1,3,4.

Figure 2. SNP-informed RNA-seq defines the male and female contributions to gene expression during reproduction. A–F. Plots of reads over gene models at representative loci. Coverage (y-axis) = log10 number of reads counted over each nucleotide. Grey color represents reads without SNPs. In regions with SNPs (green line) reads are divided into sex-specific components: Red = reads with the Cvi SNP (pistil parent), Blue = reads with the Col SNP (pollen parent). A–C, Pistil-expressed genes. D–F, Pollen-expressed genes. G–J, Genes that are abundant in pollinated pistils and are expressed in both pollen and pistil. Exons are represented by black bars, introns by black lines, and polarity of transcription is indicated by arrowheads. K. Pearson's–R correlation values are graphed with the indicated scale comparing pollen tube- and pistil-expressed genes defined by SNP-informed RNA sequencing of pollinated pistils with previously published microarray experiments (references given in text). Only genes with one unambiguous microarray ID were analyzed. Supporting bioinformatic analysis is shown in Supplemental Figure 5.

Figure 3. SNP-informed RNA sequencing defines MYB-responsive genes in the pollen tube and pistil. A. Experimental samples prepared for RNA-sequencing. B and C. Scatterplots of all average reads (ignoring SNPs) in wild-type versus myb triple-mutant pollinations. B. Differentially expressed genes identified before division of reads based upon SNPs are indicated (purple, 318 genes, adjusted p-value<0.05). C. The color code of the scatterplot (B) was altered to indicate genes with significant changes in the pollen (reads with Col SNP, light blue, N=278, p-value<0.05, DESeq2) or pistil (reads with Cvi SNP, Pink, N=34, adjusted p-value <0.05) components of their expression profile. D. A comparison of the differentially expressed genes identified with or without SNP analysis (p-value<0.05, DESeq2). E. Differential expression analysis was performed again comparing genes identified after analysis of reads without SNPs with those identified based on differential abundance (p-value <0.05, DESeq2) of pollen (Col) or pistil (Cvi) reads. Differentially expressed genes identified by both analyses are highlighted in dark colors, genes only identified by SNP-informed RNA seq are highlighted in light colors (blue, pollen reads; red, pistil reads). The total number of differentially expressed genes is shown next to each category, along with the number that were down-regulated (down) and up-regulated (up) in mutant compared to wild-type pollinations. F. Representative read coverage graphs (as in Figure 2) of MYB-dependent genes (AT1G04310 and AT1G33470). Top row - Cvi x Col-0 (wild type) pollinations, Bottom row - Cvi x myb triple-mutant pollinations. Supporting bioinformatic analysis shown in Supplemental Figure 2. Supporting bioinformatic analysis is shown in Supplemental Figure 6.

Figure 4. Pollen-specific gene expression of three MYB-regulated genes detected by SNP-informed RNA-seq. A,H,O. Sequencing reads over SNPs identifying potential MYB target genes as pollen-specific (as in Figure 2). B-F. SUC9:SUC9:GFP (AT5G06170), LAT52:DsRed. I-M. Pectate Lyase or PL:PL:GFP (AT5G09280), LAT52:DsRed. P-T. ACA4:ACA4:GFP (AT4G20990), LAT52:DsRed. B,I,P. Representative SIV pollen tube growth experiments on ms1 pistil explants, epifluorescent micrographs, scale bar = 500 µm. In each, the left image is the red channel (LAT52:DsRed) signal, which is present in the pollen grain and the tube. The right image is the green channel (GFP) signal. The autofluorescence of the ms1 stigma explant is visible in each panel. C,J,Q. Confocal micrographs of SIV-grown pollen tubes: left panel, GFP; middle panel, DsRED; right panel, merge; scale bar = 20 µm. D,K,R. Representative confocal optical sections of unpollinated pistils; the merge of DIC and GFP channels is shown. Arrows indicate expression of the GFP fusion protein in the female gametophyte, scale bar = 100 µm. E,L,S. Close up of unpollinated ovules from D,K,R. Scale bar = 100 µm, projection of red and green channels. F,M,T. Projections of confocal micrographs of pollinated pistils. Red and green channels are merged, and arrows indicate regions of strong coexpression of DsRed and the indicated GFP fusion protein (yellow) in the style. Twelve hours after pollination, scale bar = 100 µm. G,N,U. Close up of ovules targeted by GFP reporter constructs from F,M,T, scale bar = 100 µm. Supporting confocal micrographs are shown in Supplemental Figure 7; supporting RT-qPCR analysis is presented in Supplemental Figure 8.

Figure 5. Reproductive gene regulation is highly divergent between Col and Cvi. A. Hierarchical clustering of all RNA-seq samples identifies accession-specific clusters, DESeq2. B. Intersection of all expressed genes (Mean expression >100 reads) from self and inter-accession crosses. Venn diagram is not proportional to number of included elements. C. Differential gene expression observed between Col and Cvi 8 h after self-pollination. Differentially expressed genes are colored purple (adjusted p-value<0.005, DESeq2). D-F. Heat maps of Col-specific gene expression; colors represent read volume, with scales below indicating the number of reads for color value. D. Defensin-like genes. E. Low-molecular-weight cysteine-rich genes. F. Toll-interleukin receptor-nucleotide binding site-leucine rich repeat (TIR-NBS-LRR) type disease resistance proteins. G. Differential gene expression between self-pollinated Col and Cvi pistils. A total of 2,877 differentially expressed were identified following pollination, 1,640 transcripts were higher in inter-accession (Cvi x Col) pollinations, 1,238 transcripts were higher in self-pollinations (Cvi x Cvi), adjusted p-value < 0.005 (DESeq2). H. Sex-specific transcript identity of A. Red dots - Cvi-specific reads, Blue dots - Col-specific reads, and Gray dots do not have SNPs for assignment.

Figure 6. MYB transcription factors control expression of thionin CRPs (CRP2460). A. A phylogenetic tree of the CRP2460 protein coding sequences (Neighbor-joining, jukes-cantor). Two MYB-dependent members of other CRP groups (CRP2465 and CRP2530) are included for comparison. B. Expression heatmap of the CRP2460 transcripts in pistils. Cvi Unpoll – Unpollinated Cvi pistils (collected 8 h after time when pollination would have been done), Cvi x Col – Cvi pistils pollinated with Col pollen for 8 h, Cvi x myb - Cvi pistils pollinated with myb triple-mutant pollen for 8 h, Col x Col – Col pistils pollinated with Col pollen for 8, Cvi x Cvi – Cvi pistils pollinated with Cvi pollen for 8 h. Sequencing data is shown in three columns per condition, with one column per biological replicate, and color intensity is based on read number (see scale). Asterisks indicate statistically significant gene expression differences between Cvi x Col and Cvi x myb triple-mutant pollinations (adjusted p-value <0.005, DESeq2). Green plus sign (+) indicates the presence of SNPs in these CRP genes. Ka/Ks – Numbers indicate Ka/Ks ratio between CRP and homolog in A. lyrata. Gray box indicates no homolog present for this CRP. C. Representative sequencing data for two MYB-dependent CPR2460 genes, AT1G58242 and AT3G24465.

Figure 7. Elevated Ka/Ks rates in CRP genes. A. Percent of genes in indicated gene families with homologs identified in given species identified by Lloyd et al., 2015. B. Average Ka/Ks of all homologs found between Arabidopsis and the indicated species, bars are standard deviation (sample sizes vary and are defined in panel A, Lloyd et al., 2015). C. Percent of homologous gene pairs out of the total number of family members with a Ka/Ks >1 (indicator of positive selection) for each respective species. The key pertains to panels A–C.

DOI 10.1105/tpc.16.00816; originally published online April 11, 2017;Plant Cell

Alexander R Leydon, Caleb Weinreb, Elena Venable, Anke Reinders, John M. Ward and Mark A. JohnsonRNA-Sequencing

The molecular dialog between flowering plant reproductive partners defined by SNP-informed

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