Abstract
More than 80,000 angiosperm species produce flowers with petals fused into a corolla tube (i.e., sympetalous flowers). As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii. Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING 3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate up-regulation of AUXIN RESPONSE FACTOR 3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. Importantly, by integrating the molecular and phenotypic analyses of the tasiRNA-ARF pathway in Mimulus with historical insights from morphological and anatomical studies in various sympetalous species, we propose a new conceptual model for the developmental genetic control of corolla tube formation. This model offers logical connections among the sporadic previous reports of corolla tube mutants in other species and makes clear predictions that can be readily tested using the Mimulus system.
Introduction
About one third of the ∼275,000 angiosperm species produce flowers with petals fused into a corolla tube (i.e., sympetalous), forming a protective enclosure of nectaries and reproductive organs. Corolla tubes have evolved multiple times independently across the angiosperm tree of life (1), most notably in the common ancestor of the Asterids, a clade containing more than 80,000 species (2). Subsequent elaboration in length, width, and curvature has led to a great variety of corolla tube shapes that enabled asterid species to exploit many specialized pollinator groups (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation (3-6). As such, the corolla tube has long been considered a key morphological innovation that contributed to the radiation of the Asterids (1). Despite its critical importance in the reproductive success and evolution of such a large number of species, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective (7, 8).
Historically, corolla tube formation has been the subject of extensive morphological and anatomical studies (9-19). In particular, numerous studies have described the detailed ontogenetic process of corolla tube development in one subgroup of the asterid clade, the Lamiids, which contains some classical plant genetic model systems such as snapdragon (Antirrhinum), petunia (Petunia), and morning glory (Ipomoea) (11-16, 20-22). A common theme emerging from these studies is that during the early stage of petal development, petal primordia are initiated separately, followed by rapid extension of the petal bases toward the inter-primordial regions, which also grow coordinately, causing congenital “fusion” of the petal primordia and formation of the corolla tube. Little is known, however, about the genetic control of this early-phase lateral extension or the nature of the coordinated inter-primordial growth.
To date only a few genes have been implicated in corolla tube formation. Loss-of-function alleles of the FEATHERED gene in Japanese morning glory (Ipomoea nil) and the MAEWEST gene in petunia (Petunia x hybrida), both generated by transposon insertions, result in unfused corollas (23, 24). FEATHERED and encode KANADI and WOX transcription factors, and their Arabidopsis orthologs are KANADI1 and WOX1, respectively. In addition, ectopic expression of the Arabidopsis TCP5 protein fused with a repressor motif in Ipomoea also disrupted corolla tube formation (25). However, whether the endogenous TCP5 ortholog in Ipomoea is involved in corolla tube development is unclear. More recently, it was reported that transient knock-down of the Petunia NAC-transcription factors NAM and NH16 via virus-induced gene silencing (VIGS) also caused decreased petal fusion (26), but the interpretation of this result was confounded by the observation that occasional flowers produced on the “escape shoots” of the loss-of-function nam mutants have normal corolla tubes (27). The fact that these genes were characterized from different plant systems and through different methods MAEWEST (transposon insertion alleles, heterologous expression of chimeric repressor, and VIGS) makes it challenging to interpret their genetic relationships and their precise functional roles in corolla tube formation.
One way to overcome this problem is to systematically analyze corolla tube mutants in a single model system. To this end, we have employed a new genetic model system, the monkeyflower species Mimulus lewisii, mainly for its ease in chemical mutagenesis and Agrobacterium-mediated in planta transformation (28, 29). M. lewisii is a typical bumblebee-pollinated species with a conspicuous corolla tube (Fig. 1A). Through ethyl methanesulfonate (EMS) mutagenesis, we have generated a dozen recessive mutants (named flayed) with split corolla tubes. Here we report the characterization of one group of mutants, caused by loss-of-function mutations in two genes that are required for the biogenesis of trans-acting short interfering RNAs (tasiRNAs).
Among the tasiRNA loci characterized to date, TAS3 is the most widely conserved, found in virtually all land plants (30). TAS3 transcript bears two binding sites for miR390, which triggers the production of phased tasiRNAs, including the highly conserved “tasiARF” that targets AUXIN RESPONSE FACTOR 3 (ARF3) and ARF4 (31, 32). This tasiRNA-ARF regulatory module genes that are required for has been shown to play a critical role in leaf polarity and blade expansion (i.e., lamina growth) in both eudicots (33-38) and monocots (39-41). Consistent with previous studies, here we demonstrate that in the M. lewisii mutants, TAS3-derived tasiRNAs decrease dramatically in abundance and MlARF3 and MlARF4 expression are upregulated. Importantly, we show that malfunction of the tasiRNA-ARF pathway in the M. lewisii mutants impedes the early lateral expansion of the petal primordium bases and the coordinated inter-primordial growth, consequently preventing the congenital fusion of the petal primordia. Integrating our molecular and phenotypic analyses of the tasiRNA-ARF pathway in Mimulus with historical insights from morphological and anatomical studies of various sympetalous species, we propose a new conceptual model for the genetic control of corolla tube formation, which offers logical connections among the sporadic previous reports of corolla tube mutants and makes clear predictions that can be readily tested using the Mimulus system.
Results and Discussion
Phenotypic Characterization of the flayed1 and flayed2 Mutants
Three of the recessive mutants recovered from EMS mutagenesis using the inbred line LF10, flayed1-flayed3, are morphologically indistinguishable. Pair-wise crosses suggested that they belong to two complementation groups, flayed1 and flayed2 (flayed3 is allelic to flayed2) (Fig. 1B and C). In addition to having unfused petals, these mutants display carpel fusion defects, with phenotypes varying from flower to flower within the same plant. Most mutant flowers have two fused carpels, as in the wild-type, but have partially split stigmas with more than two lobes (Fig. 1E). Less frequently there are flowers with two almost completely separate styles. The length of mutant pistils is also reduced compared to the wild-type (Fig. 1E). No obvious phenotypes were observed in the stamens of these mutants.
Another notable feature of the flayed1/2 mutants is the reduced width of lateral organs. The dorsal and lateral petals show ∼30% decrease in width compared to the wild-type, and the ventral petal shows ∼37% decrease (Fig. 1F; Table S1). Leaf width is also substantially reduced (by ∼40%) in the mutants, but leaf length is unaffected (Fig. 1G and H; Table S1). To determine whether the reduction in petal width is due to change in cell number, cell size, or both, we measured the width of abaxial epidermal cells of the dorsal petal lobe for both the wild-type and the flayed2 mutant. Because the petal lobe abaxial epidermal cells are irregularly shaped (Fig. 1I), the width measurements were done on five contiguous cells to account for the variation among individual cells within the same sample. No significant difference in cell width was found between the wild-type and flayed2 (Fig. 1J), which suggests that the difference in petal width between the mutant and the wild-type is primarily due to difference in cell number (i.e., number of cell divisions).
Unlike the morning glory mutant feathered (23) or the petunia mutant maewest 24), flayed1/2 do not show any defects in tissue adaxial-abaxial polarity. Instead, the flayed1/2 mutants closely resemble the petunia mutant choripetala suzaane (chsu), which also has a split corolla tube, variable carpel fusion defects, and narrower leaf with normal adaxial/abaxial polarity. Unfortunately, the molecular identity of CHSU is still unknown.
FLAYED1 and FLAYED2 Are the Orthologs of Arabidopsis AGO7 and SGS3, Respectively
To identify the causal genes of flayed1 and flayed2, we analyzed each mutant using a genomic approach that combines the advantages of bulk segregant analysis and comparison of single nucleotide polymorphism (SNP) profiles between multiple EMS mutants (Materials and Methods), as demonstrated in a previous study (42). We narrowed the causal mutation of flayed1 and flayed2 down to 38 and 19 candidate SNPs, respectively (Table S2 and S3). The vast majority of these SNPs locate in non-coding, repetitive sequences, with only two or three mutations resulting in amino acid changes in each mutant (Table S2 and S3). Notably, in both flayed1 and flayed2, there is one mutation leading to a premature stop codon, in the ortholog of Arabidopsis ARGONAUTE7 (AGO7) and SUPPRESSOR OF GENE SILENCING 3 (SGS3), respectively (Fig. 2A and B; Table S2 and S3). AGO7 and SGS3 are part of the same tasiRNA biogenesis pathway (43-45), which would explain the indistinguishable mutant phenotypes of flayed1 and flayed2. Furthermore, sequencing the coding DNA (CDS) of MlSGS3 in flayed3, which is allelic to flayed2, revealed an independent mutation that also leads to a premature stop codon (Fig. 2B). Together, these results suggested that MlAGO7 and MlSGS3 were the most promising candidate genes for FLAYED1 and FLAYED2, respectively.
To verify gene identities, full-length CDS of MlAGO7 and MlSGS3 were introduced to the flayed1 and flayed2 mutant background, respectively, driven by the cauliflower mosaic virus 35S promoter. Among the 29 independent 35S:MlAGO7 lines in the flayed1 background, 13 showed a fully rescued phenotype that is indistinguishable from the wild-type; four lines showed a partially rescued phenotype, with petal width increased to wild-type level but the petals remained unfused (Fig. 2C). Similarly, six of the 18 MlSGS3 over-expression lines in the flayed2 background displayed a fully rescued phenotype and two displayed a partially rescued phenotype (Fig. 2D). qRT-PCR assessment of MlAGO7 and MlSGS3 expression in 5-mm floral buds showed that, in the fully rescued lines, expression levels of the transgenes are 4∼64-fold higher than those of the corresponding endogenous genes (Fig. S1). These results confirmed that MlAGO7 and MlSGS3 are indeed the causal genes underlying flayed1 and flayed2, respectively.
Knowing the causal genes and mutations allowed direct genotyping of a “flayed1 x flayed2” F2 population to identify flayed1 flayed2 double mutants, which are phenotypically indistinguishable from the single mutants (Fig. 1D, F, G, H). This further indicates that MlAGO7 and MlSGS3 function in the same genetic pathway in Mimulus, as expected.
The flayed1/2 Phenotypes Are Primarily Mediated Through the tasiRNAARF Pathway
Because AGO7 and SGS3 are necessary components of the miR390-TAS3-ARF pathway (Fig. 3A), and the highly conserved, TAS3-derived tasiARFs are known to play a critical role in leaf polarity and lamina growth by repressing ARF3/4 expression (33-41), we hypothesized that the flayed1/2 phenotypes (e.g., reduced width of lateral organs) are primarily mediated through the tasiRNA-ARF pathway. This hypothesis makes three clear predictions: (i) The abundance of TAS3-derived small RNAs, including tasiARFs, should be much lower in the mutants compared to the wild-type; (ii) The M. lewisii orthologs of ARF3/4 should be upregulated in the mutants; and, (iii) Artificial upregulation of the M. lewisii ARF3/4 orthologs in the wild-type background should recapitulate the flayed1/2 phenotypes.
To test the first prediction, we sequenced the total small RNA pool from young floral buds (5-mm) of the wild-type, flayed1, and flayed 2. Like most other angiosperms, M. lewisii has two kinds of TAS3 genes (each represented by only a single copy in the M. lewisii genome): TAS3S contains a single, centrally located tasiARF, whereas TAS3L contains two tandem tasiARFs (30; Fig. S2). No TAS3S-derived small RNAs were detected in any of the sequenced samples, suggesting that the TAS3S gene is not expressed. TAS3L-derived small RNAs were detected at the level of ∼600 per million reads in the wild-type, but decreased >50-fold in both flayed1 and flayed2 (Fig. 3B). In particular, the tasiARFs were almost entirely absent from the mutant samples (Fig. 3B). These results confirmed the first prediction.
To test the second prediction, we first searched the M. lewisii genome for ARF3/4 homologs and found a single ortholog for each of the two genes. Similar to ARF3/4 in other species, both MlARF3 and MlARF4 have two binding sites with sequences complementary to tasiARF (Fig. 4A and B). qRT-PCR measurements in 5-mm floral buds showed that in the single and double mutants, MlARF3 was up-regulated by 1.7∼2.5-fold and MlARF4 was up-regulated by 2.7∼3.7-fold (Fig. 4C). This moderate upregulation of ARF3/ARF4 in the ago7 and sgs3 mutant backgrounds is very similar to previous reports in Arabidopsis (Garcia et al., 2006; Hunter et al., 2006), supporting the role of tasiARF in fine-tuning ARF3/4 expression level.
To test the third prediction, we transformed the wild-type with a tasiARF-insensitive version of MlARF3 (mMlARF3) and MlARF4 (mMlARF4) with several synonymous substitutions at the tasiARF binding sites (Fig. 4A and B), driven by the 35S promoter. We obtained seven independent 35S:mMlARF3 and 14 35S:mMlARF4 lines. In each case, only two transgenic lines showed obvious phenotypes: their leaves are very similar to the flayed1/2 mutants (i.e., narrower than the wild-type) and corollas are partially split (indicated by the red arrow heads in Fig. 4D and E). qRT-PCR experiments on 5-mm floral buds of the transgenic lines with even the strongest phenotypes showed only moderate overexpression of MlARF3/4 relative to the wild-type (2∼4-fold, Fig. S3A and B). Examination of two random 35S:mMlARF4 lines without obvious phenotypes showed no increase in expression level of MlARF4 (Fig. S2C). The lack of 35S:mMlARF3/4 lines with strong transgene expression is in contrast to ectopic expression of MlAGO7 and MlSGS3 (Fig. S1) as well as pigment-related transcription factors in M. lewisii (46, 47), where the same 35S promoter could readily drive transgene expression level >10-fold higher than that of the endogenous genes. One possible explanation for this observation is that transgenic lines with very strong ARF3/4 expression in M. lewisii are seedling lethal, as implicated by similar experiments in tomato (37). Nevertheless, our results show that a moderate up-regulation of MlARF3/4 can indeed fully recapitulate the leaf phenotype and partially recapitulate the flower phenotype of the flayed1/2 mutants. Furthermore, a double transgenic line derived from a cross between the strongest 35S:mMlARF3 line and 35S:mMlARF4 line showed dramatic petal fusion defects (Fig. 4F), more closely resembling the flayed1/2 mutants than the single transgenic lines. This indicates that MlARF3 and MlARF4 may act synergistically in regulating corolla tube formation. Taken together, our results from transgenic manipulation of the MlARF3/4 expression levels suggest that the flayed1/2 phenotypes (narrow leaf and split corolla tube) are primarily mediated by the up-regulation of MlARF3/4.
The tasiRNA-ARF Pathway Is Required for Preferential Lateral Expansion of the Bases of Petal Primordia and Coordinated Growth of Inter-primordial Regions
To understand how malfunction of the tasiRNA-ARF pathway affects corolla tube formation in M. lewisii, we have studied floral organogenesis in the wild-type and the flayed2 mutant using scanning electron microscopy. Like other species in the Lamiid clade (e.g., snapdragon, petunia, morning glory), M. lewisii petals are initiated as five separate primordia (Fig. 5A). Petal development lags behind stamens in the early stages (Fig. 5B and C), but by the time the corolla reaches ∼0.5 mm in diameter (Fig. 5D), petal development progresses rapidly and soon the stamens are found enclosed in the corolla (Fig. 5E-H). The developmental stage from 0.3 to 0.4 mm (corolla diameter) is critical for corolla tube formation: during this stage, the bases of the petal primordia quickly expand laterally (to a conspicuously greater extent than the upper portion of the petal primordia; Fig. 5M), and the inter-primordial regions also grow coordinately, connecting the initially separate petal primordia. Floral organogenesis of flayed2 is very similar to that of the wild-type at the early stages (before the corolla reaches 0.3 mm in diameter; Fig. 5I). However, during the critical period (0.3∼0.4 mm), there is no preferential lateral expansion at the bases of the petal primordia, manifested as the truncate shape of the petal primordium base (Fig. 5N), in contrast to the semi-circle shape of the wild-type (Fig. 5M). Notably, growth of the inter-primordial regions is also arrested in flayed2, leading to a gap between two adjacent petal primordia (Fig. 5J-L and indicated by the asterisk in Fig. 5N).
Given that disruption of tasiRNA biogenesis and the consequent up-regulation of ARF3/4 have been shown to cause reduced lamina growth of lateral organs in multiple plant species (36-38, 41), it is not surprising to observe reduced lateral expansion at the bases of the petal primordia in flayed2 compared to the wild-type (Fig. 5M and N). But how does this relate to the arrest of upward growth of the inter-primordial regions?
In a series of careful anatomical studies of various taxa in the Asterid clade, Nishino (12-15) recognized that the “co-operation” between the marginal meristem activities of the base of the petal primordia and the upward growth of the inter-primordial regions plays a pivotal role in corolla tube formation, although the nature of this “cooperation” was unclear. In light of this earlier insight, we interpreted the flayed2 phenotype as follows: in the wild-type there is molecular signaling from the marginal meristematic cells at the bases of the petal primordia to the inter-primordial cells, stimulating the latter to grow upward coordinately (i.e., the nature of the “co-operation” is molecular signaling between the two regions). In the flayed2 mutant, this molecular signaling is disrupted, causing a complete arrest of the upward growth of the inter-primordial regions. An obvious candidate for this putative signal is the phytohormone auxin, which is known to promote localized tissue outgrowth and meanwhile suppress organ boundary genes such as CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 in Arabidopsis (48).
A Conceptual Model for the Genetic Control of Corolla Tube Formation
Two recent attempts of building a conceptual framework for floral organ fusion in general (7) or petal fusion in particular (8) both emphasized the genetic regulatory network underlying organ boundary formation and maintenance. The rationale for such emphasis was explicitly stated by Specht and Howarth (7): “fusion as a process may more accurately be defined as a lack of organ separation”. While these attempts represent an important step towards a mechanistic understanding of the developmental process of corolla tube formation, to some degree they have neglected the insight provided by earlier morphological and anatomical studies (i.e., the importance of the “cooperation” between the rapid lateral expansion of the petal primordium bases and the upward growth of the inter-primordial regions), and have not provided a logical integration of the sporadic reports of corolla tube mutants (23, 24, 49).
Building on the historical insight from anatomical studies and our molecular and phenotypic analyses of the tasiRNA-ARF pathway in Mimulus, we propose a new conceptual model that offers logical connections among the sporadic previous reports on the genetic control of corolla tube formation (Fig. 6). This model consists of three interconnected modules. At the heart of the model is the molecular signaling from the marginal meristematic cells at the base of the petal primordia to the inter-primordial cells, providing a molecular explanation for the “co-operation” between the petal primordia and inter-primordial regions observed in anatomical studies. Upstream of this core module is the genetic regulatory network responsible for lateral expansion of the petal primordium base, which is required for initiation of the molecular signaling. Downstream of this core module lie the organ boundary genes that would suppress localized tissue growth if not repressed by the molecular signal coming from the petal primordia.
This model can readily explain the phenotypes of loss-of-function mutations in the morning glory FEATHERED gene and the petunia MAEWEST gene, which encode KANADI and WOX transcription factors, respectively (23, 24). Together with the tasiRNA-ARF pathway, these transcription factors are part of the genetic network regulating leaf adaxial-abaxial polarity and lamina growth (50-52). According to our model, disrupting this genetic regulatory network is expected to impede lateral expansion of the petal primordia, as in the flayed2 mutant (Fig. 5N), and consequently break the signaling from the petal primordia to the inter-primordial cells, resulting in unfused petals. A less dramatic corolla tube phenotype was also observed in the snapdragon graminifolia mutant, with fusion defect restricted to the two dorsal petals (49). GRAMINIFOLIA encodes a YABBY transcription factor (49), another component of the leaf polarity/lamina growth genetic network. This example further highlights the importance of the “lamina growth” module in the model (Fig. 6).
Our model also provides a plausible explanation for the petal fusion defects observed when a chimeric repressor of AtTCP5 was over-expressed in the Japanese morning glory (25). Chimeric repressors of CIN-like TCP transcription factors, including TCP5 in Arabidopsis, are known to activate organ boundary genes such as CUC1/2 (53). Ectopic activation of CUC1/2 is expected to prevent the upward growth of the interprimoridial regions (i.e., boundary between adjacent petal primordia). Also consistent with the “organ boundary” module (Fig. 6) is a recent observation in snapdragon –– the expression of the CUC ortholog, CUPULIFORMIS, is cleared from the inter-primordial regions shortly after petal initiation but later is reactivated in the sinuses between adjacent corolla lobes (54). Through computational modeling, Rebocho et al. (54) showed that this “gap” of CUPULIFORMIS expression (between the base of the corolla and the sinuses) is necessary for corolla tube formation.
In addition to explaining these previous observations, our model predicts that transgenic manipulation of other components of the leaf polarity/lamina growth network (e.g., AS1, AS2, HD-ZIPIII) (50-52) or ectopic expression of organ boundary genes (e.g., CUC, ORGAN BOUNDARY1, JAGGED LATERAL ORGAN) (55-57) in sympetalous species may also result in unfused corollas; it also predicts that misregulation of the molecular signaling, as proposed in the core module (Fig. 6), in transgenic or mutant plants should produce defective corolla tubes. If the putative signal is indeed auxin, regulation of polarized auxin transport within and between petal primordia will be key to understanding corolla tube formation. The availability of multiple corolla tube mutants, the ease of bulk segregant analysis to identify mutant genes, and the amenability of Agrobacterium-mediated in planta transformation make Mimulus a favorable system to test these predictions and to dissect the detailed molecular mechanisms and developmental process of corolla tube formation.
Materials and Methods
Plant Materials and Growth Conditions
EMS mutagenesis was performed using the Mimulus lewisii Pursh inbred line LF10 as described (28). Another inbred line SL9 was used to generate the “flayed x SL9” F2 populations. Plants were grown in the University of Connecticut research greenhouses under natural light supplemented with sodium vapor lamps, ensuring a 16-hr day length.
Phenotypic Characterization
To quantify phenotypic differences between the mutants and wild-type, we measured the widths of the dorsal, ventral, and lateral petals using a digital caliper. We also measured the lengths and widths of the fourth leaf (the largest leaf) of mature plants. To further evaluate whether the width difference is due to change in cell number, cell size or both, width of the abaxial epidermal cells of the dorsal petal lobe was measured following a previously described procedure (58).
Genomic Analyses for Causal Gene Identification
To identify causal genes underlying flayed1 and flayed2, we employed a hybrid strategy that combines the advantages of bulk segregant analysis and genome comparisons between multiple EMS mutants, as described previously (42). Briefly, for each mutant an F2 population was produced by crossing the homozygous mutant (generated in the LF10 background) and the mapping line SL9. DNA samples from 96 F2 segregants displaying the mutant phenotype (i.e., homozygous for the causal mutation) were pooled with equal representation. A small-insert library was then prepared for the pooled sample and was sequenced using an Illumina HiSeq 2000 platform at the University of North Carolina High Throughput Sequencing Facility. About 213 and 448 million 100-bp paired-end reads were generated for flayed1 and flayed2, respectively.
The short reads were mapped to the LF10 genome assembly version 1.8 (http://monkeyflower.uconn.edu/resources) using CLC Genomics Workbench 7.0 (Qiagen, Valencia, CA). The causal mutation should be: (i) homozygous for the pooled sample (i.e., 100% SNP frequency in the “F2 reads – LF10 genome” alignment); and (ii) unique to each mutant (i.e., any shared 100% SNPs between mutants are most likely due to assembly error in the reference genome or non-specific mapping of repetitive sequences). After comparisons to the SNP profiles of previously published mutants, guideless (59), rcp1 (47), act1-D (58), and rcp2 (60), we narrowed the causal mutation to 39 and 19 candidate SNPs for flayed1 and flayed2, respectively (Fig. S3).
Small RNA Sequencing and Analyses. For small RNA sequencing, total RNA was first extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich) from 5-mm floral buds of LF10, flayed1, and flayed2 (two biological replicates for each genotype). Small RNA libraries were then constructed using the TruSeq Small RNA Sample Preparation Kits (Illumina), with the total RNA as starting material. The libraries were sequenced on an Illumina HiSeq 2500 at the Delaware Biotechnology Institute (Newark, DE). Small RNA reads were quality-controlled and adaptor-trimmed before calculating tasiRNA abundance, as described in Xia et al. (30).
Quantitative RT-PCR
RNA extraction and cDNA synthesis were as previously described (29). cDNA samples were diluted 10-fold before quantitative reverse transcriptase PCR (qRT-PCR). All qRT-PCRs were performed using iQ SYBR Green Supermix (Bio-Rad) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Samples were amplified for 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Amplification efficiencies for each primer pair were determined using critical threshold values obtained from a dilution series (1:4, 1:8, 1:16, 1:32) of pooled cDNA. MlUBC was used as a reference gene as described (29). Primers used for qRT-PCR are listed in Table S4.
Plasmid Construction and Plant Transformation
To generate the 35S:MlAGO7 and 35S:MlSGS3 constructs for the rescue experiments, we first amplified the full-length CDS of MlAGO7 and MlSGS3 from the wild-type LF10 cDNA using the Phusion enzyme (NEB, Ipswich, MA). For each gene, the amplified fragment was cloned into the pENTR/D-TOPO vector (Invitrogen) and then a linear fragment containing the CDS flanked by the attL1 and attL2 sites was amplified using M13 primers. This linear fragment was subsequently recombined into the Gateway vector pEarleyGate 100 (61), which drives transgene expression by the CaMV 35S promoter. To generate the 35S:mMlARF3/4 constructs, CDS of insensitive forms of MlARF3 (mARF3) and MlARF4 (mARF4) that carries synonymous substitutions in the two tasiRNA recognition sites were synthesized by GenScript (NJ, USA) and then cloned into the pEarleyGate 100 destination vector as described for the 35S:MlAGO7 and 35S:MlSGS3 constructs. All plasmids were verified by sequencing before being transformed into Agrobacterum tumefaciens strain GV3101 for subsequent plant transformation, as described in Yuan et al. (29). Primers used for plasmid construction and sequencing are listed in Table S5.
Scanning Electron Microscopy
Flower buds were fixed overnight in Formalin-Acetic-Alcohol (FAA) at 4°C, dehydrated for 30 min through a 50%, 60%, 70%, 95%, and 100% alcohol series. Samples were then critical-point dried, mounted, and sputter coated before being observed using a NOVA NanoSEM with Oxford EDX at 35 kV at UConn’s Bioscience Electron Microscopy Laboratory.
Author Contributions
B.D. designed the study, performed most of the phenotypic characterization and functional experiments, analyzed and interpreted the data, and drafted the manuscript. R.X. and B.C.M. acquired and analyzed small RNA data. V.G. and P.K.D. acquired and interpreted the SEM images. J.M.S., L.E.S., and M.S. contributed to the transgenic experiments. Q.L. interpreted the data and illustrated the model (Figure 6). Y.W.Y. conceived and designed the project, analyzed and interpreted the data, and drafted the manuscript. All authors reviewed the manuscript.
Data Availability
Short read data have been deposited in NCBI SRA (BioProject PRJNA423263); small RNA data have been deposited in NCBI GEO (GSE108530); annotated gene sequences have been deposited in GenBank (MG669632- MG669634 and MF084285).
Acknowledgements
We are grateful to Dr. Toby Bradshaw (University of Washington) for encouragement and initial support for generating the bulk segregant data in his laboratory. We thank Clinton Morse, Matt Opel, and Adam Histen for plant care in the UConn EEB Research Greenhouses. This work was supported by the University of Connecticut start-up funds and an NSF grant (IOS-1558083) to Y-W.Y., and an NSF grant (IOS-1257869) to B.C.M.