Variation in the expression of a transmembrane protein influences cell growth in Arabidopsis thaliana petals by altering auxin availability

Background The same species of plant can exhibit highly diverse sizes and shapes of organs that are genetically determined. Defining genetic variation underlying this morphological diversity is an important objective in evolutionary studies and it also helps identify the functions of genes influencing plant growth and development. Extensive screens of mutagenised Arabidopsis populations have identified multiple genes and mechanisms affecting organ size and shape, but relatively few studies have exploited the rich diversity of natural populations to identify genes involved in growth control. Results We screened a relatively well characterised collection of Arabidopsis thaliana ecotypes for variation in petal size. Association analyses identified sequence and gene expression variation on chromosome 4 that made a substantial contribution to differences in petal area. Variation in expression of At4g16850 (named as KSK), encoding a hypothetical protein, had a substantial role on variation in organ size by influencing cell size. Over-expression of KSK led to larger petals with larger cells and promoted the formation of stamenoid features. The expression of auxin-responsive genes known to limit cell growth was reduced in response to KSK over-expression. ANT expression was also reduced in KSK over-expression lines, consistent with altered floral identities. Auxin availability was reduced in KSK over-expressing cells, consistent with changes in auxin-responsive gene expression. KSK may therefore influence auxin availability during petal development. Conclusions Understanding how genetic variation influences plant growth is important for both evolutionary and mechanistic studies. We used natural populations of Arabidopsis thaliana to identify sequence variation in a promoter region of Arabidopsis ecotypes that mediated differences in the expression of a previously uncharacterised membrane protein. This variation contributed to altered auxin availability and cell size during petal growth.


Keywords
Arabidopsis thaliana/organ size variation/natural genetic variation/auxin responses Background Cell proliferation and cell growth are coordinated to generate the characteristic sizes, shapes and functions of plant organs. This coordination involves multiple cellular processes, including signalling mechanisms, cell division, turgor-driven cell expansion, and cell wall and protein synthesis [1]. During the formation of determinate plant organs such as leaves and petals, cell proliferation with limited cell growth occurs at earlier stages of organ formation, followed by cell growth with limited cell proliferation occurs to increase cell size, accompanied by differentiation as the developing organ attains its final characteristic size and shape [2]. Very little is known about the spatial and temporal integration of cell proliferation and cell growth to generate the final sizes and shapes of organs and seeds, despite its fundamental and applied importance.
Many plant species display a wide range of forms due to altered sizes and shapes of organs, reflecting adaptation to their natural environments. The natural range of the annual species Arabidopsis thaliana extends from northern Scandinavia to Africa, and it exhibits a correspondingly diverse range of phenotypes [3][4][5], such that most ecotypes are phenotypically distinct. However, genetic variation underlying this phenotypic variation is still poorly described.
For example, the extent to which variation in the functions of genes influencing organ size established in one widely studied ecotype influences natural variation in organ sizes in populations of Arabidopsis thaliana is not well understood. Also, the extent of conservation of known mechanisms influencing organ size and many other traits in natural populations is also insufficiently documented. Therefore, an increased understanding of the genetic foundations of natural variation in traits such as organ size may help to both identify mechanisms and to shed light on how natural genetic variation influences organ size and other traits.
Arabidopsis thaliana has adapted to diverse habitats worldwide and extensive natural variation in organ size reflects these different life histories [4]. Although variation in the shapes and sizes of different floral organs are correlated in order to maintain the reproductive functions of the flower [6], significant genetic variation influencing floral morphology traits was identified by QTL analyses of Arabidopsis Recombinant Inbred Line (RIL) populations [7,8]. More recently, QTL analyses identified six independent loci influencing variation in petal shape and size, with variation at the ERECTA (ER) locus accounting for 51% of this variation [9]. Haplotype variation in 32 ecotypes at a known locus, GA1, was associated with variation in petal, stamen and style lengths [10]. In one of the few studies exploiting natural variation to identify BRX as a regulator of cell proliferation during root growth [11]. Despite these studies, there are few studies that have characterised the functions of natural variation in organ size in Arabidopsis.
Genome-wide association (GWA) mapping in Arabidopsis is increasingly used to access a wider range of natural genetic variation, to identify small-effect alleles, and to map genotypephenotype relationships more precisely [12]. The very small size of its genomes has facilitated the re-sequencing of a large range of Arabidopsis thaliana ecotypes and the identification of vast numbers of SNP and small indel variants by comparison to the assembled Col-0 ecotype [13]. Within this comprehensive set of ecotypes, those from Sweden are relatively well documented [14] and have been screened for variation in over-wintering responses [15]. Initial inspection of this collection showed considerable variation in petal size and shape, therefore we conducted an association analysis of 272 sequenced Swedish ecotypes. We identified variation in the promoter of a previously undescribed gene, At4G16850, encoding a predicted 6transmembrane (6TM) protein. Ecotypes and transgenic lines with increased At4g16850 expression had larger petals due to increased cell growth. Over-expression of At4g16850 reduced expression of several auxin-responsive genes that reduce petal cell size, and also reduced auxin availability. At4g16850 over-expression also led to the partial homeotic conversion of petals to stamenoid structures, and this was attributed to altered expression of floral organ development genes.

Results
Identifying a locus influencing petal area.
We measured the length, maximum width and area of petals of 272 Arabidopsis thaliana ecotypes collected from southern and northern Sweden (Additional file 1) that were grown in controlled conditions after vernalization. Additional file 2 shows the petal phenotype data. All three petal parameters varied substantially within the sampled collection. For example, mean petal areas varied from 0.915 mm 2 (Hov1-10) to 4.92 mm 2 (Vår2-6), a difference of 537%.
Additional file 3 shows representative petals from these ecotypes and from Död 1, an intermediate size for comparison. Figure 1A shows that mean petal area variation formed a normal distribution and was therefore suitable for association studies. GWAPP was used [16] with an Accelerated Mixed Model, incorporating information across 250,000 SNPs. This analysis identified a significant SNP association on chromosome 4 for petal area ( Figure 1B). The most significantly associated SNP within this region was located at position 9471419 bp, within gene model At4G16830. The marker at this position was bi-allelic, with those ecotypes carrying an "A" allele at this locus exhibiting a ~15% increase in petal area relative to those carrying the alternative "T" allele ( Figure 1C). The extent of Linkage Disequilibrium (LD) in the region was six Arabidopsis gene models, from At4g16820 to At4g16850. Sequence variation altering the activities of these genes may explain the variation in petal size observed across ecotypes.
Assessment of gene annotations revealed no known regulators of petal or organ size. The effect of genetic variation within the haplotype defined by LD on petal growth was assessed in a subset of three ecotypes with small petal areas and three ecotypes with large petal areas ( Figure 1E). Petal cell areas and numbers were quantified using Scanning Electron Microscopy (SEM) and Image J. A significant increase in petal abaxial epidermal cell area was observed in ecotypes carrying the increasing A allele at position 9471419 relative to ecotypes carrying the decreasing T allele ( Figure 1E). Therefore, the major effect of genetic variation in the haplotype is on petal cell area.
Expression levels of At4g16850 are correlated with quantitative variation in petal size.
To assess the potential role of the 6 candidate genes in regulating petal growth, petal areas were measured in available potential loss-of-function mutants in the ecotype Col-0. T-DNA insertion lines were available from stock centres for all genes found to be in high LD with associated markers with the exception of At4g16850, a small gene of unknown function. We measured the expression of these 6 candidate genes in developing floral tissues in the six ecotypes with varying petal sizes. For At4g16820 to At4g16845 no differences in petal area were seen in the T-DNA insertion lines relative to Col-0 plants ( Figure 2C). Furthermore, no differential expression of these genes in developing flowers was observed between the six ecotypes with small and large petals ( Figure 2B).  Figure 2B). relatively small petal phenotype. Col-0 was therefore selected as the "small petal" reference genome due to the high level of sequence conservation between small petal ecotypes and Col-0. However, no full-length promoter amplicon could be generated from any of the large petal ecotypes. We therefore generated whole genome assemblies using Illumina sequence of unamplified DNA templates [17] made from the three large petal ecotypes to access sequence variation in At4g16850. An ABYSS de novo assembly generated a large contig spanning the  Supplementary Figure 2 shows the sequence alignment of the intergenic regions.
B. The coding regions of At4g16850 from the large petal ecotype Dju-1 and the small petal ecotype Col-0 were aligned to identify predicted protein sequence differences. The coding regions were analysed with InterPro to identify putative transmembrane and cytoplasmic protein domains, shown as coloured bars under the predicted coding sequence. Amino acid differences are shown as gray highlights.
C. Transient expression from the 35S promoter of At4g16850 coding region fused to GFP at its C-terminus in Col-0 petal protoplasts. A known plasma-membrane protein TMK4 fused to RFP was used to reveal co-location in the plasma membrane. The white colour in the overlay reveals co-location of At4g16850-GFP and TMK4-RFP at the plasma membrane.
At4g16850 encodes a predicted 6-transmembrane domain protein with 3 non-cytoplasmic domains and 4 cytoplasmic domains ( Figure 3B). Comparison of the Dju-1 and Col-0 assemblies revealed the predicted protein was highly conserved between these large-and small-petal ecotypes, with only two non-conservative amino acid changes in trans-membrane region 4 and in the C-terminal cytoplasmic domain ( Figure 3B). To assess the predicted subcellular location of the protein encoded by At4g15850, its coding region was fused at its Cterminus with GFP and transiently expressed from the 35S promoter in Col-0 developing petal protoplasts, together with a known transmembrane receptor-like kinase TMK5 [18] fused to RFP. Confocal imaging showed that the At4g16850-GFP fusion protein co-localised with the RFP-tagged TMK5 plasma membrane protein ( Figure 3C). The At4g16850-GFP fusion protein was also observed in cytoplasmic structures.
Overexpression of At4G16850 increases petal size due to increased cell growth Correlation analysis of the expression of At4G16850 across ecotypes displaying high variation for petal area established that its differential expression explained 76% of the variation in petal size in the analysed ecotypes ( Figure 4A).
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Previous studies have identified several genes that influence petal cell growth in Arabidopsis.
BPEp [19] and ARF8 function together [20] to limit petal cell growth, and FRL1 [21] also represses petal cell growth. The expression of these genes in developing petals of three transgenic Col-0 lines over-expressing At4g16850 and in untransformed Col-0 was measured using Q-RT-PCR to assess whether KSK may influence petal cell growth through these genes.
Although only one transgenic line showed significant reduction in BPE expression in petals  Overexpression of KSK leads to partial homeotic conversion of petals to stamenoid structures.
In addition to observing a significant increase in petal cell growth in the 35S::KSK overexpressing lines, we also observed partial organ identity changes in ~10% of flowers from all eight 35S::KSK transgenic plants. These flowers, in addition to the expected four petals in the second whorl, carried a 5th petal-like structure. This developed in the outer margin of the second whorl and displayed stamenoid features such as a partial pollen sac ( Figure 6A) and stomata, a cell type not observed in Col-0 petals ( Figure 6B). The presence of stomata on petal epidermal surfaces has also been seen in ant mutants deficient in the transcription factor AINTEGUMENTA (Krizek 2000). Using qRT-PCR, we assessed ANT expression in developing petals of the three 35S::KSK over-expressing lines. A significant decrease in ANT expression was observed in petals overexpressing KSK ( Figure 6C). This suggests that KSK expression levels contribute to determining floral organ identity in a pathway involving ANT.
Reduced auxin responses in lines over-expressing KSK.
One common feature of ARF8 and FRL1 expression, which was suppressed by over-expression of KSK, is that the expression of both genes increases in response to auxin [20,23,24]. We therefore tested auxin responses in petals of wt Col-0 and hrs to allow for protein expression, protoplasts were treated with either 0 nM or 1000 nM IAA.
Protoplasts were imaged between 1-2 hr after IAA treatment. Figure 6D shows the nuclear localisation of both fluorophores and Figure 6E shows relative fluorescence of mDII-Tomato/DII-Venus measured in the nuclei of transfected petal protoplasts. The increased ratio in Col-0 protoplasts shows a reduction in DII-Venus protein compared to mDII-Tomato in response to auxin, demonstrating that transiently-expressed protoplasts respond to added auxin similarly to stable transgenic plants [25]. In contrast, in 35S::KSK transgenic protoplasts, the ratio of Venus to Tomato fluorescence was not decreased to the same extent as Col-0. This indicated that auxin responses may be reduced in this transgenic line. This interpretation was tested by measuring the expression levels of two auxin-responsive genes (IAA1 and IAA9) in seedlings of Col-0 and KSK over-expressing lines. Their expression was reduced ( Figures 6F,G), supporting the interpretation that auxin responses are decreased in KSK over-expressing lines.

Discussion
Approximately 7,000 Arabidopsis thaliana ecotypes have been systematically collected and 1,000 of these have been sequenced to access a wide range of genomic diversity [13]. We used genome-wide association analyses [26] on a set of sequenced ecotypes collected from Sweden [14] to identify a major source of sequence variation influencing petal size. Variation in the expression of a gene encoding a hypothetical plasma membrane protein, which we call KSK, made a major contribution to variation in petal size in this set of ecotypes. Auxin responses as measured by the R2D2 reporter were reduced in KSK over-expression lines, suggesting that the KSK membrane protein may directly or indirectly influence auxin responses or levels in developing petals.
KSK was predicted to encode a transmembrane protein with 6 short helical domains spanning the membrane, 3 non-cytoplasmic domains and 4 cytoplasmic domains in an N-in conformation ( Figure 3B). A KSK-GFP fusion protein was co-localized with a plasma membrane protein ( Figure 3C). The KSK protein sequence is reasonably highly conserved among several groups of plants, and it has no close family members in Arabidopsis, with only very partial protein alignments to At1g31130 detected by reciprocal BLASTP searches. At1g31130 is a 321aa predicted 6TM protein located in Golgi, endosomes and the plasma membrane. The 6TM protein structure is predicted to be present in many Arabidopsis proteins with diverse functions, including aquaporins, voltage-gated ion superfamily transporters, and mitochondrial carrier proteins [27]. The MIND1 database of membrane protein interactions [28] identified an interaction between KSK and At1g07860, encoding a predicted Receptor-Like Cytoplasmic Kinase VII (RLCK VII) family member. Members of this family function in pathogen-triggered immunity and growth pathways [29] and include BIK1, which is membrane anchored via Nmyristoylation [30]. In KSK over-expressing lines, auxin responses were reduced as detected by transient expression of the R2D2 auxin reporter gene ( Figure 6E). This may be due to reduced auxin responses, synthesis, or altered transport. The membrane localisation of KSK-GFP suggests a potential influence on auxin transport. However, the predicted 6TM transmembrane organisation of KSK in membranes is different from that of all known auxin uptake and efflux plasma membrane-and tonoplast-located auxin transporters [31].
Multiple promoter polymorphisms were identified between the small petal ecotype Col-O and the large petal ecotype Dju-1 ( Figure 3A and Additional Figure 2) after re-sequencing and assembling Dju-1. In contrast, the protein coding regions of these two ecotypes had only two non-conserved amino acid changes ( Figure 3B) Polymorphisms of this length are very common in Arabidopsis genome assemblies [32], and are over-represented in many eukaryotic genomes, where they may be generated by replication slippage [33]. dA:dT tracts in promoters have a well-established role in regulating gene expression by forming part of scaffold attachment regions (SARS) and by introducing curvature in DNA that influences transcription factor and nucleosome access.
KSK over-expression led to reduced expression of ARF8 and FRL1, two genes that exert a specific negative effect on petal cell size ( Figures 5A,B,C). FRL1 encodes a sterol methyltransferase that influences endoreduplication [34]. ARF8 is an auxin-responsive transcription factor that forms a transcription complex with the bHLH transcription factor BPEp [20]. BPEp also restricts cell expansion specifically in petals. BPEp is highly expressed during the later stages of petal development, while ARF8 is ubiquitously expressed, but more highly expressed during the later stages of petal development. The expression of both FRL1 and ARF8 is increased in response to auxin [23,24,35], and as auxin responses are reduced in KSK over-expressing lines ( Figure 6E), it is possible that KSK over-expression may reduce expression of these auxin-responsive negative regulators of petal cell size, leading to increased petal cell size and overall increases in petal area. The down-regulation of ANT expression in KSK over-expression lines ( Figure 4C) is consistent with reduced auxin responses, as auxin increases ANT gene expression [36]. ANT encodes an AP2/ERF transcription factor that influences several stages of floral development, including specification of floral organ identity [37]. Petals in KSK over-expressing lines often exhibited a partial conversion to stamenoid features, and also had stomata, a cell type not normally found in petals ( Figures 6A, B). In ant mutants, petal cell identity was also altered to form stomata [22], supporting the interpretation using OligoDT. All protocols were carried out using manufacturers' guidelines. cDNA samples were diluted 1:10 in water before use. Q-RT-PCR was carried out using SYBR green real time PCR mastermix (Thermofisher) and performed using Lightcycler 480 (Roche, Switzerland).
Primer sequences used for q-RT-PCR are in Additional file 5. Primer efficiencies and relative expression calculations were performed according to methods described by [42]. All q-RT-PCR assays were repeated at least twice. All PCR reactions were carried out using Phusion High Fidelity DNA polymerase (New England BioLabs) according to manufacturer's instructions.
Capillary sequencing was carried out by GATC Biotech (Germany). For whole genome assembly of ecotypes Dja-1, TBA_01 and TI_070, high MW DNA was prepared and PCR-free indexed Illumina libraries prepared as described [43]. After QC approximately 50m 150bp paired-end reads were generated (Novagene, Hong Kong) for each library. Cleaned reads were assembled using ABySS v1.3.6 [44] with a k-mer of 75. Genome assemblies were aligned with the genomic region of At4g16850 using MUSCLE v3.8.31 [45]. Assemblies of the three ecotypes are available at ENA (PREJB28030).

Transient expression in Arabidopsis protoplasts
Transient expression assays were carried out using protoplasts isolated from Arabidopsis Col-0 developing petals [46]. Protoplasts were transformed with 5ug plasmid DNA (purified using the