Expression analysis of the MIR172 family in long days

The expression patterns of the MIR172 genes were previously assessed by semiquantitative reverse transcription PCR (RT-PCR) in whole seedlings [22]; however, the expression of these genes in the SAM was not determined. Therefore, we harvested RNA from apical tissue of LD-grown plants through a developmental time course and performed quantitative reverse transcription PCR (RT-qPCR; Fig 1A–1F). We analyzed the accumulation of the MIR172 precursor RNAs, because the mature forms encoded by each precursor are too similar to distinguish by this method [8]. Under these conditions, the floral transition occurs between 14 and 17 days after germination, as defined by the level of AP1 transcript (Fig 1A) and microscopic examination (see later). MIR172A was more highly expressed at 7 days, was at trough levels at 10 days, and then increased again through 12, 14, and 17 days (Fig 1B). In contrast, MIR172B and MIR172D RNAs were at trough levels during vegetative development, but increased during floral transition (Fig 1C–1E). Thus, the abundance of these 3 MIR172 RNAs increased most sharply at the apex during the floral transition and was closely correlated with AP1 expression, although significant increases in MIR172A RNA were observed prior to AP1 expression (Fig 1B). MIR172C and MIR172E were very lowly expressed in samples derived from vegetative plants or plants undergoing the floral transition; however, MIR172C was expressed at 21 days (Fig 1D–1F).

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Fig 1. Expression patterns of the MIR172 gene family in the shoot apex.

(A–F) Graphs depicting RT-qPCR results of (A) AP1, (B) MIR172A, (C) MIR172B, (D) MIR172C, (E) MIR172D, and (F) MIR172E expression in materials derived from the shoot apex of LD-grown plants that were harvested at the indicated times after germination. Expression was normalized to 7d samples. Error bars indicate SEM of 3 independent biological replicates. Data underlying panels A–F are provided in S1 Data. (G, H) Confocal laser scanning micrographs of the shoot apices of (G) MIR172A-NVG, (H) MIR172B-NVG, and (I) MIR172D-NVG transgenic plants grown in LD conditions and harvested at the indicated times after germination. (H) Note fluorescence in L1 at 21d MIR172B-NVG^11.3 meristem (inset, arrowheads). Fluorescence from the Venus protein is artificially colored in green, and the fluorescence from the Renaissance dye is artificially colored in magenta. LD, long-day; NVG, NLS-Venus-GUS; RT-qPCR, quantitative reverse transcription PCR; SEM, standard error of the mean.

https://doi.org/10.1371/journal.pbio.3001043.g001

To improve the resolution of the spatial expression patterns, we cloned the 5 intergenic regions containing the MIR172A-E genes and replaced the miR172 hairpin regions with the coding region for a nuclear localized fusion between the fluorescent protein Venus and β-glucuronidase (NLS-Venus-GUS, NVG) (S1 Fig). Notably, the size and genomic structure of each of the MIR172 genes varied greatly (S1 Fig), which might contribute to their diverse expression patterns. We analyzed several independent transformants of each construct and selected at least 2 for more detailed analysis (Fig 1, S2 and S3 Figs). Apical tissue of these lines was harvested at the same time points described for the RT-qPCR experiments and analyzed by confocal microscopy (Fig 1, S2 and S3 Figs). Only MIR172A-NVG, MIR172B-NVG, and MIR172D-NVG were reproducibly expressed at the shoot apex with each of their temporal expression patterns largely in agreement with the RNA profiling study (Fig 1G–1I, S2A–S2C and S2F Fig). Expression of MIR172A-NVG and MIR172D-NVG was detected from around day 14 in and below the L3 layer of the SAM (Fig 1G–1I, S2A–S2F Fig), whereas MIR172B-NVG expression was mainly detected in the rib meristem and the vasculature extending from the main shoot to floral buds (Fig 1H, S2B and S2C Fig). Fluorescence was also observed in the L1 of MIR172B-NVG (Fig 1H–21d, S2B and S2C and S3D Figs) and MIR172D-NVG (Fig 1I—17–19d, S2F Fig) transgenic plants, although this occurred only at specific times. MIR172C-NVG was mainly expressed in the vasculature of the inflorescence stem (S2D and S2E and S3G–S3I Figs); however, we were unable to detect expression of MIR172E-NVG in the shoot apex. Taken together, these data demonstrate that the MIR172 genes exhibit diverse temporal and spatial expression patterns and that MIR172A, MIR172B, and MIR172D increase in expression in or near the shoot apex before and during the floral transition, whereas MIR172C is mainly expressed in the inflorescence stem, and MIR172E activity could not be detected in the apex of LD-grown plants.

Inactivation of all MIR172 genes using genome editing

Null or strong loss-of-function mutants for the MIR172 genes are not available in A. thaliana, although alleles that reduce the activity of MIR172A and MIR172D have been described [6,34]. Therefore, we generated mutations in each of the 5 MIR172 genes using previously described CRISPR-Cas-9 systems [52,53]. We inactivated the MIR172 genes by deleting or severely disrupting the sequences that comprise the miRNA/miRNA* duplex or by disrupting the sequences that are necessary for DICER-LIKE 1 (DCL1)-mediated processing [15,16,54]. The guide RNAs used, their activity, and the sequences of the stably inherited mutations are shown in S4 Fig. We identified 3 classes of mutation: (i) modifications of the stem region beneath the miRNA/miRNA* duplex (mir172a-2, mir172a-3, mir172b-1, and mir172b-3); (ii) modifications of the miRNA/miRNA* duplex (mir172d-2 and mir172d-3); and (iii) deletions of the entire miRNA region or the entire miRNA/miRNA* duplex (mir172c-1 and mir172e-1) (Fig 2A, S4M Fig). Notably, the predicted secondary structures of the first class of mir172 mutant precursors were altered relative to their WT counterparts (S5 Fig), suggesting that their processing would be impaired [15,16]. To test the effect of the mutations, we ectopically expressed the endogenous and mutant genes in planta to assay their effects on flowering time. While the mean total leaf number (TLN) produced by plants ectopically expressing the WT MIR172 genes was lower than the empty vector control, the mean TLN produced by plants expressing mutated versions was similar to the empty vector control (Fig 2B, Table 1, S1 Table). These data indicated that the mir172 mutant alleles were strongly impaired in function and may be null alleles, and this was further supported by testing mir172 levels in higher-order mutants (see below).

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Fig 2. Functional analysis of single mir172 mutants LDs and SDs.

(A) Schematic depicting the structure of a representative “reference” miR172 precursor and the mutant forms generated using CRISPR-Cas-9 DNA editing. The orange, red, and green bars represent the coding sequences for miR172, miR172*, and the biogenesis site required for DCL1 cleavage, respectively. Truncation of these bars and opaque sections of the mutant mir172 precursor indicates the regions that were deleted via CRISPR-Cas-9 in mir172a-2, mir172b-3, mir172c-1, mir172d-2, and mir172e-1. (B) A graph depicting the TLN of first generation transformants in a Col-0 background harboring a T-DNA containing only the UBQ10pro-3′OCS promoter-terminator cassette or expressing reference Col-0 or mutated versions of the indicated MIR172 gene under the control of the UBQ10pro-3′OCS cassette. (C, D) Graphs depicting the TLN and bolting times of WT and plants harboring single mir172 mutations in (C) LD and (D) SD conditions. Data underlying panels B–D are provided in S2 Data. (E–G) Confocal laser scanning micrographs of the shoot apices of (E) MIR172A-NVG, (F) MIR172B-NVG, and (G) MIR172D-NVG transgenic plants grown in SD conditions and harvested at the indicated times after germination. Fluorescence from the Venus protein is artificially colored in green, and the fluorescence from the Renaissance dye is artificially colored in magenta. Cas-9, CRISPR associated protein-9; CRISPR, clustered regularly interspaced short palindromic repeats; LD, long-day; NVG, NLS-Venus-GUS; SD, short-day; TLN, total leaf number; WT, wild-type.

https://doi.org/10.1371/journal.pbio.3001043.g002

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Table 1. Flowering time experiments and statistical groupings.

https://doi.org/10.1371/journal.pbio.3001043.t001

Analysis of flowering time behavior of mir172 mutants

We removed the T-DNA harboring the CRISPR-Cas-9 cassette by genetic crossing and generated quintuple mutants using a selection of these novel mir172 alleles. We performed whole genome sequencing of the resulting quintuple mutant and found no off-target mutations induced by the CRISPR-Cas-9 cassettes (Materials and methods). We re-isolated all individual single mutants from plants that were segregating for each selected mir172 mutant and performed flowering time assays in long and short photoperiods. Of the single mutants grown under LD conditions, the average TLN of mir172a-2 and mir172b-3 were reproducibly higher relative to the reference WT genotype, Col-0 (Fig 2C, Table 1, S1 Table). In days to bolting (DTB), mir172a-2 was slightly higher than Col-0 (Fig 2C, Table 1, S1 Table). By contrast, these metrics were mostly comparable between the other mir172 mutant alleles and Col-0 (Fig 2C, Table 1, S1 Table). Therefore, MIR172A and MIR172B are the most important in floral promotion under LDs.

Under SD conditions, mir172a-2 also flowered later than Col-0, whereas mir172b-3 and mir172c-1 flowered at a time similar to Col-0 (Fig 2D, Table 1, S1 Table). In contrast to LD conditions, mir172d-3 flowered much later than Col-0 when grown under SD conditions (Fig 2D, Table 1, S1 Table). These results suggested that miR172 activity is more important in the absence of inductive photoperiods and that MIR172D contributes to a large degree of this activity. Therefore, we tested the expression of our MIR172 reporters under these conditions (Fig 2E–2G, S6 Fig) and found that the expression patterns in the shoot apex were similar to those observed under LDs. However, expression of MIR172A-NVG was not detectable until 2 to 3 weeks of growth under SDs, compared with approximately 10d under LDs (Fig 2E, S6A Fig). Expression of MIR172B-NVG and MIR172D-NVG was also not detectable until 3 weeks of growth in SDs (Fig 2F and 2G, S6B, S6C–S6F Fig), compared to 10 to 12 LDs.

We then analyzed the flowering time behavior of mir172 mutant combinations and observed progressively later flowering under both LD and SD conditions as more MIR172 genes were inactivated (Fig 3A and 3B). The mir172abcd quadruple mutant flowered later than the mir172abc triple mutant in both conditions, but it was substantially later under SDs, consistent with an important role for MIR172D under these conditions (Fig 3B, Table 1, S1 Table). The leaf number and DTB were comparable between quintuple mir172abcde mutant plants and quadruple mir172abcd mutant plants that retained a functional MIR172E allele, consistent with a weak role for this gene in controlling flowering time (Fig 3B). Furthermore, both quadruple and quintuple mutants grown under short photoperiods initially flowered from axillary shoots rather than the primary shoot (70/77 plants), indicating that miR172 is essential for the earlier timing of the floral transition at the SAM compared to axillary meristems under these conditions (Fig 3C). Siliques of quadruple and quintuple mutant plants exhibited a swollen shape, indicative of defective floral meristem termination (S7A Fig). The severity of this phenotype decreased acropetally, which mirrored previous reports of the severity of ap2 single mutant phenotypes increasing acropetally [55]. Plants lacking MIR172A, MIR172B, and MIR172D activity also bore severely serrated cauline leaves (S7B Fig), which were less serrated in mir172abc or mir172ab mutants and very similar to Col-0 in the single mutant combinations. Supported by the imaging and RT-qPCR results presented above, these results suggest that MIR172E plays a very minor role during the floral transition and floral organ development.

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Fig 3. Flowering time of higher-order mir172 mutants in LDs and SDs.

(A, B) Graphs depicting DTB and TLN or RLN of Col-0, mir172a-2, mir172a-2 b-3, mir172a-2 b-3 c-1, mir172a-2 b-3 c-1 d-3, and mir172a-2 b-3 c-1 d-3 e-1 mutant combinations in LD (A) and SD (B) conditions. The difference between the means of the indicated genotypes is shown above the box plots. Data underlying panels (A) and (B) are provided in S2 Data. (C) Photographs of mir172a-2 b-3 c-1 d-3 e-1 plants after approximately 6 months of growth under SD conditions. Plant initiated flowering shoots from axillary meristems (white arrowheads) rather than the primary meristem. (D, E) Graphs depicting DTB and total or RLN of Col-0, mir172a-2 b-3 d-2, mir172a-2 b-3 c-1 d-3 e-1 mutant combinations and 35Spro:MIM172 in LD (D) and SD (E) conditions. Data underlying panels (D) and (E) are provided in S2 Data. (F) A photograph of Col-0, mir172a-2 b-3 d-2, mir172a-2 b-3 c-1 d-3 e-1 mutant combinations and 35Spro:MIM172 grown under SD conditions. DTB, days to bolting; LD, long-day; RLN, Rosette leaf number; SD, short-day; TLN, total leaf number.

https://doi.org/10.1371/journal.pbio.3001043.g003

With the exception of the comparison between quadruple and quintuple mutants, the smallest effect size in terms of leaf number and DTB was observed when comparing mir172abc and mir172ab mutants (Fig 3A and 3B, Table 1, S1 Table). The TLN and DTB of mir172c-1 e-1 double mutants were similar to Col-0 under LDs (S7C Fig, Table 1, S1 Table). Furthermore, all mir172abc plants grown under SD conditions flowered from the main shoot and bore siliques of WT appearance. This suggests that although MIR172C may play some role in controlling flowering time, its contribution is small when compared to MIR172A, MIR172B, and MIR172D.

The TLN and DTB of mir172ab were clearly higher than Col-0 in both photoperiods (Fig 3A and 3B, Table 1, S1 Table). Although the differences between mir172a single mutants and Col-0 were similar to our previous results (Fig 2C and 2D, Table 1, S1 Table), the variation was increased in these experiments (Fig 3A and 3B, Table 1, S1 Table). We noted that the number of leaves produced by mir172a mutants often appeared greater relative to Col-0 than the differences in DTB. Therefore, we tested the leaf initiation rate of mir172 mutants and found that the plastochron was longer than Col-0, with robust differences arising after 40 d of growth under short photoperiods (S7D Fig). Therefore, counting the leaf number of mir172 mutants alone is not sufficient to assess their flowering times.

Taken together, these genetic data supported the idea that MIR172A, MIR172B, and MIR172D are the main regulators of flowering time in the MIR172 gene family. This is in agreement with the expression patterns of the reporter constructs, with only MIR172A-NVG, MIR172B-NVG, and MIR172D-NVG being detectably expressed in the shoot apex prior to and around the time of the floral transition. To test this further, we produced mir172a-3 b-3 d-2 triple mutants and compared their flowering time behaviors under long and SDs with the mir172abcde quintuple mutants generated above and the previously described 35Spro:MIM172 transgenic line, which ectopically expresses a target mimicry that depletes miR172 activity [5] (Fig 3D–3F). As observed previously, mir172abcde plants flowered much later than Col-0 (Fig 3D–3F). The leaf numbers and DTB of mir172abd were very similar to mir172abcde under both conditions (Fig 3D and 3E, Table 1, S1 Table), with most mir172abd and mir172abcde plants initially flowering from axillary shoots when grown under short photoperiods. The leaf numbers and DTB of 35Spro:MIM172 transgenic lines were lower than both the triple and quintuple mutants under LDs and SDs (Fig 3D and 3E, Table 1, S1 Table). Furthermore, the morphology of the 35Spro:MIM172 rosette leaves was markedly different to any of the mir172 mutant plants, and the rosette diameter was substantially smaller, which was particularly noticeable in SDs (S7F Fig). We conclude that MIR172A, MIR172B, and MIR172D are the primary regulators of flowering time in the MIR172 family. We also conclude that miR172 action is essential for flowering from the SAM under noninductive photoperiods and strongly delays flowering, while under inductive photoperiods, it plays a less important role in flowering, and this can be eventually bypassed.

Activity of miR172 in the leaves and meristems under inductive photoperiods

Under inductive LD photoperiods, gene activity in leaves and shoot apices make major contributions to floral induction; therefore, to understand the roles of the MIR172 genes under these conditions, we examined the expression of the MIR172-NVG reporters in the leaves of plants grown under LDs. For this analysis, we used histological staining with the β-glucuronidase enzyme (GUS), which comprises part of our NVG reporters (S1, S8 and S9 Figs). Strong GUS enzyme activity was detected in the cotyledons and throughout the leaves of MIR172B-NVG plants, which grew weaker as the leaf aged. In contrast, MIR172A-NVG activity was weakly but reproducibly detected in the vasculature of young late-arising rosette and/or cauline leaves (approximately 21 d), and MIR172C-NVG was sometimes detected in a similar pattern (Fig 4, S8 and S9 Figs). MIR172D-NVG expression was not reliably detected in leaves (Fig 4, S8 and S9 Figs). These patterns are largely in agreement with a small RNA sequencing (RNA-seq) dataset in which the abundance of the miR172a-b isoform was 3 orders of magnitude higher in the leaves than the miR172c-d or miR172e isoforms (S9O Fig) [56].

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Fig 4. Activity of MIR172 genes in leaves.

(A–C) Photographs of 14-day-old (A) MIR172A-NVG, (B) MIR172B-NVG, and (C) MIR172D-NVG transgenic plants grown in LD conditions after GUS staining and clearing. Scale bar indicates 2 mm. (D) Graphs depicting RT-qPCR results of miR172 (upper panel) and FT mRNA (lower panel) in leaves 1 and 2 of Col-0, mir172b-3 and mir172a-2 b-3 c-1 d-3 e-1 grown for 12 LDs, harvested at ZT 16. Each point represents an individual biological replicate. Each biological replicate was normalized to the mean of the 3 Col-0 biological replicates. Data underlying panels D are provided in S1 Data. (E) Micrographs of RNA in situ hybridizations using a commercial probe designed to recognize miR172 of Col-0 (upper panels) and mir172a-2 b-3 c-1 d-3 e-1 (lower panels) apices grown under LD conditions for the indicated times. FT, FLOWERING LOCUS T; GUS, β-glucuronidase enzyme; LD, long-day; NVG, NLS-Venus-GUS; RT-qPCR, quantitative reverse transcription PCR; ZT, zeitgeber time.

https://doi.org/10.1371/journal.pbio.3001043.g004

Together, these data indicate that MIR172B makes a major contribution to the pool of miR172 present in the leaves. To quantify the amount of miR172 present, we performed RT-qPCR on mature miR172 in Col-0, mir172b single mutants, and mir172abcde quintuple mutants. MIR172B contributed 50% to 73% of total miR172 levels in leaves 1 and 2 of 12-day-old LD-grown plants (2-tailed paired t test p = 0.021). We did not test miR172 levels in the other single mutants, but in leaves of mir172abcde, quintuple mutants miR172 levels were at approximately 1% of those in Col-0 (2-tailed paired t test p = 0.009) (Fig 4D). This quantitative result is consistent with the absence of miR172 signal in the shoot apices of quintuple mutant plants, as determined by RNA in situ hybridization both under LDs and SDs (Fig 4E, S7E Fig). Some of the AP2-LIKE TFs whose mRNAs are miR172 targets are thought to directly suppress the transcription of the florigen-encoding FT gene in leaves [7,22,27]. We therefore tested FT mRNA levels in the same leaf material of these genetic backgrounds and found that FT mRNA levels were between 49% and 79% of Col-0 levels in mir172b mutants (2-tailed paired t test p = 0.008) (Fig 4D). In mir172abcde quintuple mutants, FT mRNA levels were between 27% and 31% of Col-0 levels (2-tailed paired t test p = 0.015), which suggests that reduced FT partially contributes to the late-flowering phenotype of LD-grown mir172 quintuple mutants (Fig 4D). However, sufficient FT and TSF expression may be retained to facilitate some response to LDs, as suggested by the dramatic difference between miR172abcde mutant flowering in LD- and SD-grown plants (Fig 2A and 2B).

Interplay of AP2 and MIR172 during the floral transition

MiR172 inhibits the activity of AP2-like proteins either by inducing degradation of their mRNAs or inhibiting their translation [8,22,45]. Unfortunately, the activities and the mRNA or protein expression profiles of most of these AP2-LIKE genes have not been precisely determined. Furthermore, two of the best characterized AP2-LIKE genes, TOE1 and SMZ, are genetically linked to MIR172A and MIR172D, respectively, complicating genetic analysis (S10 Fig). Moreover, little is known about TOE2 and SNZ activity, and the main function of TOE3 is probably during floral organ formation [24]. Therefore, we initially focused on testing the relationship between AP2 and miR172 during floral transition. Previous work that included genome-wide assessments of AP2 activity suggested it exerts the majority of its influence on flowering time at the shoot apex [21], although it appears to be expressed throughout the plant [57]. Furthermore, RNA in situ hybridization showed that AP2 mRNA is highly abundant in the vegetative SAM but reduced in the inflorescence meristem [31], which is consistent with its role as a negative regulator of flowering time. To understand how the expression pattern of AP2 progresses during the floral transition, we generated a fluorescently tagged version of AP2 driven by its endogenous regulatory sequences (S11 Fig). We then assessed the expression pattern of AP2-Venus in Col-0 by confocal laser microscopy under LDs (Fig 5A). AP2-Venus was present in the shoot meristem at 14 days after germination but at later time points, as the floral transition proceeded, AP2-VENUS was depleted from the apex. This pattern therefore was anticorrelated with the expression of the MIR172-NVG reporters (Fig 1). We then introgressed the AP2-VENUS transgene into the mir172abd triple mutant background and monitored the expression of AP2-VENUS in LDs (Fig 5A). AP2-VENUS was not detected in the mir172abd plants at 14 days after germination, but the signal appeared at 17 LDs and became strongly expressed in a broader domain at 19 LDs (Fig 5A). At 21 LDs, AP2-VENUS was no longer detected at the shoot meristem. This experiment indicated that in mir172abd plants the expression of AP2-VENUS protein at the shoot apex is delayed and that it then persists later in development than in Col plants. Interestingly, similar observations were made through in situ hybridizations with AP2 mRNA, as it was absent from the apices of WT plants by 16 LDs but persisted in mir172abcde mutants until at least 22 days (Fig 5B). To provide a broader view of the effect of mir172abd mutations on the abundance of the mRNAs of AP2 and the AP2-L genes, we performed an RNA-seq analysis of apices of Col-0 and mir172abd plants grown under LDs and SDs (Fig 5C and 5D, S11 Fig, S3 Table). At this level of resolution, the abundance of the mRNAs of AP2 and most AP2-L genes in mir172abd plants was similar to Col-0 throughout the time course. However, the mRNA of TOE2 was significantly reduced in Col-0 compared to mir172abd at later time points under LDs and SDs. This result is consistent with a previous analysis that found that in plants overexpressing miR172, TOE2 mRNA was strongly reduced, while the mRNA levels of AP2 and other AP2-L genes were not significantly altered, perhaps due to impaired feedback regulation [45].

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Fig 5. AP2 activity in the shoot apex.

(A) Confocal laser scanning micrographs of the shoot apices of AP2-Venus transgenic plants grown in LD conditions in a reference Col-0 background (upper panels) and mir172a-2 b-3 d-2 background (lower panels). Fluorescence from the Venus protein is artificially colored in green, and the fluorescence from the Renaissance 2200 dye is artificially colored in magenta. (B) Micrographs of RNA in situ hybridizations using a commercial probe designed to recognize AP2 mRNA of Col-0 (upper panels) and mir172a-2 b-3 c-1 d-3 e-1 (lower panels) apices grown under LD conditions for the indicated times. (C, D) Graphs depicting RNA-seq-derived data of the mRNA levels in apices of AP2, TOE2, and AP1 in Col-0 and miR172abd at the indicated time points after germination in (C) LDs and (D) SDs. Error bars represent the standard deviation of 3 biological replicates. Data underlying panels (C) and (D) are provided in S1 Data. (E, F) Graphs depicting the DTB and T/RLNs of Col-0, ap2-12, miR172a-2 b-3 d-2, and mir172a-2 b-3 d-2 ap2-12 plants grown under (E) LD and (F) SD conditions. Data underlying panels (E) and (F) are provided in S2 Data. (G) Photograph of Col-0, ap2-12, mir172a-2 b-3 d-2, and ap2-12 mir172a-2 b-3 d-2 plants grown in LD conditions. AP2, APETALA2; DTB, days to bolting; LD, long-day; RLN, Rosette leaf number; SD, short-day; TLN, total leaf number.

https://doi.org/10.1371/journal.pbio.3001043.g005

The extended duration of expression of AP2-VENUS in mir172abd plants indicated that the persistence of AP2 in the SAMs of mir172abd plants might contribute to their later flowering. To test this directly, we introduced the null ap2-12 allele into mir172abd triple mutants and scored their flowering time under long and short photoperiods (Fig 5E and 5F). As previously reported, ap2-12 produced fewer leaves and bolted earlier than Col-0 under both LDs and SDs (Fig 5E–5G, Table 1, S1 Table) [21]. Consistent with this, the number of leaves and bolting time of ap2 miR172abd quadruple mutants were much reduced compared to miR172abd triple mutants under both LDs and SDs (Fig 5E–5G, Table 1, S1 Table). Although inactivation of AP2 in the mir172abd mutant produced earlier flowering plants, the quadruple mutant bore more leaves and bolted later than Col-0 (Fig 5E and 5F, Table 1, S1 Table). These results demonstrate that ectopic and/or prolonged AP2 activity contributes to the late-flowering phenotype of mir172abd mutants and that ectopic expression of other AP2-LIKE genes, including TOE2 (Fig 5C and 5D), likely participates in repressing the floral transition of mir172abd mutants under LDs and SDs.

Synergistic functions of MIR172 and FUL during floral transition

In the mature inflorescence, the MADS-box TF FUL binds to the regulatory regions of AP2, SNZ, and TOE1 to repress their transcription [47], whereas miR172 represses these genes at the posttranscriptional level. Whether this dual mechanism of repression of AP2-LIKE gene expression occurs during floral transition is unclear. Therefore, we tested the effect of combining ful-2 and mir172 mutations on flowering time (Fig 6A, S12A and S12B Fig).

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Fig 6. Interplay between Venus-rSPL15, MIR172, and FUL.

(A) Graphs depicting DTF and TLN between combinations of ful-2 and mir172a-2 b-3 c-1 d-3. Data underlying panel A are provided in S2 Data. (B, D) Photographs of (B, C) flowers arising from ful-2 mir172a-2 b-3 c-1 d-3 plants and (D) an approximately 4-month-old ful-2 mir172a-2 b-3 c-1 d-3 plant. (E–J) RT-qPCR results of (E) MIR172A, (F) MIR172B, (G) MIR172C, (H) MIR172D, (I) MIR172E, and (J) FUL expression in materials derived from the shoot apex of LD-grown Col-0 and rSPL15-Venus plants that were harvested at the indicated times after germination. Expression was normalized to 7 d Col-0 samples. Error bars indicate SEM of 3 independent biological replicates. Data from Col-0 is the same as in Fig 1, but tissue collection was originally paired with rSPL15-Venus. (K, L) Graphs depicting DTB and T/RLN between the indicated combinations of rSPL15-Venus, mir172a-2, miR172b-3, and ful-2 mutants grown in (K) LD and (L) SD conditions. Data underlying panels (K) and (L) are provided in S2 Data. DTF, days to the first flower opening; FUL, FRUITFULL; LD, long-day; RLN, Rosette leaf number; RT-qPCR, quantitative reverse transcription PCR; SD, short-day; SEM, standard error of the mean; TLN, total leaf number.

https://doi.org/10.1371/journal.pbio.3001043.g006

We constructed the ful-2 mir172a-2 b-3 c-1 d-3 quintuple mutant and compared its flowering time under LDs with Col-0 and parental controls (Fig 6A, Table 1, S1 Table). As previously observed, ful-2 had a higher cauline leaf number (CLN) and the days to the first flower opening (DTF) was higher than Col-0 under these conditions, although the TLN of ful-2 and Col-0 was similar (Fig 6A, Table 1, S1 Table). The quintuple ful-2 mir172a-2 b-3 c-1 d-3 mutants bore more leaves and opened the first flower later than mir172a-2 b-3 c-1 d-3 (Fig 6A, Table 1, S1 Table). These flowering time data indicated a synergistic genetic interaction when compared to the expected additive interaction between ful-2 and mir172a-2 b-3 c-1 d-3 [Δ18.08 TLN (p < 10–5), Δ3.45 DTF (p = 0.002)].

Under SD conditions, ful-2 was later flowering than Col-0 with slightly fewer rosette leaves, but more cauline leaves (S12B Fig, Table 1, S1 Table), in agreement with previous reports [58]. Under these conditions, the ful-2 mir172a-2 and ful-2 mir172b-3 double mutants were scored due to the strong late-flowering phenotype of the ful-2 mir172a-2 b-3 c-1 d-3 quintuple mutant in LDs (Fig 6A). The ful-2 mir172b-3 plants flowered substantially later than ful-2 mir172a-2 or the single mutant progenitors under SDs (S12B Fig, Table 1, S1 Table), which supports a synergistic interaction between FUL and MIR172B under these conditions. Notably, under LDs, the relative severity of the double mutant flowering time was reversed compared to SDs, so that the ful-2 mir172a-2 double mutant flowered later than ful-2 mir172b-3 with similar leaf numbers (S12A Fig, Table 1, S1 Table). This result suggests a photoperiod-dependent input into different MIR172 genes. Taken together, these data support a redundant role for FUL and miR172 in controlling flowering time. This genetic redundancy probably involves the suppression of AP2-LIKE activity through transcriptional and posttranscriptional means, as was observed in mature inflorescences [47].

In further support of redundancy between FUL and mir172 in regulating AP2-LIKE genes in different developmental contexts, flowers of ful-2 mir172a-2 b-3 c-1 d-3 plants lost meristem determinacy to a greater degree than mir172 quintuple mutants (Fig 6B and 6C, S7A Fig), while ful mutants do not develop indeterminate flowers [47,48]. Furthermore, the longevity of ful-2 mir172a-2 b-3 c-1 d-3 was extended far beyond mir172a-2 b-3 c-1 d-3 or ful-2, with plants continuing to develop flowers after 5 months of growth under LDs (Fig 6D). These phenotypes are similar to those described for plants ectopically expressing miR172-resistant mRNAs of AP2-LIKE TFs, which delay flowering, increase inflorescence meristem activity, and reduce floral meristem determinacy [7,8,24,27,47].

MIR172 and FUL contribute to SPL15 function during floral transition

SPL15 is an important regulator of flowering time under SDs, although it also plays a minor role under LD conditions that is circumvented by the photoperiod pathway [35,41]. Notably, miR172 and the mRNA of FUL are undetectable by in situ hybridization in the shoot apices of spl15 mutants grown under SDs for 9 weeks, whereas in WT plants, both FUL mRNA and miR172 are detectable from 5 weeks [35]. Furthermore, SPL15 binds to the regulatory regions of MIR172B and FUL, indicating that these genes are direct targets of SPL15 [35]. To test further the idea that SPL15 activates transcription of MIR172 genes and FUL, we tested the levels of MIR172A-E RNAs and FUL mRNA in a background containing an SPL15 transgene controlled by its endogenous regulatory regions and engineered to be resistant to miR156 (Venus-rSPL15) (Fig 6E–6J) [35]. FUL and all MIR172 genes, except MIR172E, were expressed at higher levels than in Col-0. We then combined Venus-rSPL15, mir172a-2 b-3, and ful-2 and tested their flowering behaviors under both long and SDs (Fig 6K and 6L). Control experiments using Venus-rSPL15 and Col-0 plants showed that Venus-rSPL15 bolted earlier after germination and produced fewer leaves than Col-0 (Fig 6K and 6L, Table 1, S1 Table). However, the time between germination and the DTF was similar for both genotypes (S12C Fig). Thus, the time between DTB and DTF was extended in the Venus-rSPL15 genotype relative to Col-0, which was also reflected in the longer stem length of Venus-rSPL15 compared to Col-0 at the time DTF (S12C Fig). The introgression of either ful-2 or mir172a-2 b-3 into Venus-rSPL15 delayed flowering time. This delay was enhanced further in a Venus-rSPL15 ful-2 mir172a-2 b-3 background (Fig 6K and 6L, Table 1, S1 Table). However, Venus-rSPL15 ful-2 mir172a-2 b-3 still flowered earlier than the ful-2 mir172a-2 b-3 triple mutant. These data provide genetic support for MIR172AB and FUL acting downstream of SPL15, although SPL15 must also activate independent factors that can promote flowering in the absence of MIR172AB and FUL activity.

Late flowering of spl15 mutant is strongly enhanced by mir172 or ful mutations

To determine whether MIR172 and FUL only act downstream of SPL15 or whether they also have independent functions during floral transition, we constructed genotypes carrying the loss-of-function spl15-1 mutation as well as ful-2 or mir172a-2 and mir172b-3, and measured their flowering times (Fig 7A, S12D and S12E Fig).

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Fig 7. Genetic interactions between mir172, ful, and spl15 mutations.

(A) A box plot depicting DTB for plants carrying the indicated combinations of spl15-1, ful-2, and mir172 mutations and grown in SD conditions. Data underlying panel A are provided in S2 Data. (B) A bar chart depicting the proportion of plants from (A) that flowered from the SAM or axillary meristems. The data for combinations between ful-2, mir172a-2, and mir172b-3 are supplemented by data from S12B Fig. Data for mir172abcd(e/+) are taken from Fig 3B and are shown here for comparison. The total number, N, of plants analyzed is indicated. Data underlying panel B are provided in S2 Data. (C) Schematic to summarize the interactions coordinated by SPL15 during the floral transition. Red lines with flat caps indicate negative regulation, and blue arrows indicate positive regulation. Gray text and line color indicate inactivity at that stage of development. Black text indicates activity at that stage of development. AP2-LIKE, APETALA2-LIKE; DTB, days to bolting; FUL, FRUITFULL; SAM, shoot apical meristem; SD, short-day; SPL, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE.

https://doi.org/10.1371/journal.pbio.3001043.g007

Under LD conditions, the flowering times of spl15-1 mir172a-2 b-3 and spl15-1 ful-2 were delayed relative to Col-0 and the parental genotypes (S12D and S12E Fig). The interaction between spl15-1 and mir172a-2 b-3 appeared to be largely additive when compared with the expected additive interaction [Δ3.16 TLN (p = 0.13), ΔDTF (p = 0.94)] (S12D Fig). This result is consistent with MIR172A and MIR172B retaining activity in LD-grown spl15-1 mutants, and therefore that AP2-LIKE genes are down-regulated at least partially independently of SPL15 under these conditions [35,41]. The interaction between spl15-1 and ful-2 appeared to be synergistic when compared to the expected additive interaction [Δ7.73 TLN (p = 0.0001), Δ3.05 DTF (p = 0.0001)] (S12E Fig). Given that FUL is required for complete FT activity [49,59], the loss of FUL function in LDs may enhance the importance of SPL15 when the photoperiod pathway is partially disrupted, which indicates that FUL and SPL15 do not act in a simple linear pathway under LDs.

Under SD conditions, many of the genotypes produced extremely high numbers of leaves, so we recorded only the number of days from sowing to bolting for many genotypes in a large-scale greenhouse experiment (Fig 7A), although we also performed smaller-scale experiments with combinations of ful-2, mir172a-2, and mir172b-3 in growth cabinets, as described above (S12B Fig). Each of the parental genotypes behaved as previously observed as spl15-1 bolted much later than Col-0, mir172a-2 bolted slightly later than Col-0, and mir172b-3 bolted at a similar time to Col-0 (Fig 7A, Table 1, S1 Table). The spl15-1 mir172a-2 double mutant bolted much later than spl15-1 and spl15-1 mir172b-3 bolted even later, with both interactions appearing to be synergistic compared to the expected additive interaction [Δ22.73 DTB; p = 0.0002] and [Δ35.48 DTB; p < 10–5], respectively (Fig 7A, Table 1, S1 Table). This result indicated that MIR172A and MIR172B activities are retained in SD-grown spl15-1 mutants and promote flowering, although miR172 abundance is reduced in spl15-1 [35].

Under SDs, ful-2 also bolted later than Col-0, as expected (Fig 7A, Table 1, S1 Table). The spl15-1 ful-2 double mutant bolted extremely late compared to spl15-1 in a clearly synergistic interaction compared to the expected additive interaction (Δ33.78 DTB; p < 10−5) (Fig 7A, Table 1, S1 Table). This experiment also indicated that FUL activity is retained in spl15-1, although its expression is reduced and delayed [35]. Taken together, these data indicate that although MIR172 and FUL are transcriptionally activated by SPL15, they retain activity in spl15-1 mutants, and mutations in MIR172 genes or FUL strongly enhance the spl15 mutant phenotype.

Flowering of primary shoots is more dependent on SPL15, FUL, and MIR172 than flowering of axillary shoots

Several genotypes analyzed under SDs were severely impaired in floral transition at the SAM and initiated flowering from axillary branches, although most of these plants did eventually flower from the SAM as well; in contrast, WT Col-0 always flowered first from the SAM (Fig 7B). Under SDs, 14% (2/14) spl15-1 and 14% (2/14) ful-2 mir172b-3 plants initiated flowering from axillary meristems at the same time or before the SAM (S13A and S13B Fig). However, all ful-2 mir172a-2 (14/14) or ful-2 mir172a-2 b-3 (14/14) plants flowered first from the SAM. In an independent experiment, 13% (6/46) of spl15-1 and 20% (3/15) of ful-2 mir172b-3 plants bolted from axillary meristems at the same time or before the SAM. Once again, all ful-2 mir172a-2 (36/36) and ful-2 mir172a-2 b-3 (34/34) plants flowered from the SAM before the axillary meristems, as observed with WT Col-0 plants. Thus, in this respect, the ful-2 mir172b-3 double mutant phenocopied spl15-1. Furthermore, the proportion of plants that bolted from axillary shoots first was higher in spl15-1 mir172a-2 (21%, 6/28), spl15-1 mir172b-3 (68%, 19/28), and spl15-1 ful-2 (54%, 6/11) compared to spl15-1 alone. This phenotype appears to be driven by an increase in AP2-LIKE activity as determined by the high proportion of mir172 quadruple or quintuple mutants that flowered first from axillary meristems (91%, 70/77). These data from all experiments are summarized in Fig 7B. Furthermore, although this loss of apical dominance is more severe in short photoperiods, when these mutant combinations were grown in LD conditions, they exhibited similar architectural defects (S13D–S13H Fig). These results indicate that transcriptional activation of FUL and MIR172 by SPL15 may be essential for flowering of the SAM in some plants and that these genes ensure the temporal progression of flowering from the shoot apex to axillary branches during shoot growth.

Essential activity of miR172 at the shoot apex

Flowering time analysis of mir172 quintuple mutants combined with gene expression profiling showed that MIR172A, MIR172B, and MIR172D are the primary regulators of flowering time at the shoot apex (Figs 1 and 2). The product of MIR172B appears to also account for at least half of the total miR172 pool in the leaves (Fig 4D), but other MIR172 genes are also expressed in leaves (S8 Fig). Although miR172 increases FT expression indirectly, this regulation does not control the photoperiod response in a binary manner. Rather, it appears that miR172 modulates the response to photoperiod, perhaps as a means to provide developmental robustness, as has been observed for miRNAs in metazoans [60]. Such a role would explain the observation that MIR172 expression appears to overlap with AP2-LIKE expression in the leaves and cotyledons rather than being mutually exclusive [7,22,57], although understanding their functions in leaves will require more thorough investigation at the tissue and cellular levels. Responses to other environmental stimuli, such as ambient temperature and winter cold [61], may be modulated by miR172 in a similar manner.

At the shoot apex, expression of MIR172 and AP2, one of the 6 members of the AP2-LIKE family, appears to be mutually exclusive (Fig 5A). AP2 mRNA and protein is rapidly cleared from the shoot apex during floral transition, which is probably important for a robust flowering response. This mechanism is particularly important under noninductive photoperiods as the photoperiod pathway appears to largely bypass the requirement for miR172 activity under inductive photoperiods. Genome-wide transcriptome profiling also showed that the mRNA of TOE2 is highly increased under SDs in the mir172abd mutant background compared to Col-0, suggesting that TOE2 also contributes to late flowering of the mutant under these conditions. TOE2 mRNA was previously shown to be reduced by overexpression of miR172, whereas the mRNA of AP2 and other AP2-L genes was not [45]. It was proposed that these TFs feedback to repress their own transcription [21,45], so that reductions in their protein levels by overexpression of miR172 may lead to higher levels of their mRNAs. This feedback regulation together with cross-regulation among different members of the family makes it difficult to predict the temporal expression levels of AP2-L proteins in the miR172 mutants. Indeed, we detected an unexpected delay in AP2-Venus accumulation in the apices of mir172abd mutants. Expression and genetic analysis of the whole AP2-L family will be required to fully decipher the interactions among these proteins.

Loss of MIR172A-E or MIR172A, MIR172B, and MIR172D delayed flowering under noninductive photoperiods by approximately 3 months compared to Col-0 controls. Furthermore, once these mir172 mutants did flower, they mostly did not do so from the primary SAM but from axillary meristems, although usually the primary shoot did eventually flower. This loss of apical dominance suggests that MIR172 activity is required to confer a coherent spatial response to inductive flowering cues and not only for timing the transition. Inactivation of AP2 partially suppressed the late flowering of mir172abd plants, although the ap2 mir172abd plants still flowered much later than Col-0 (Fig 5B and 5C). The effects of introducing mutations in other AP2-L genes, particularly TOE2, into mir172abd mutants remain to be tested. Our RNA-seq analysis suggests that several of these are expressed at the shoot apex and may also be important in controlling the floral transition at the shoot apex. Notably, Zhang and colleagues showed that toe1 and toe1 toe2 plants are extremely early flowering under both SD and LD conditions.

Transcriptional and posttranscriptional regulation of AP2-LIKE activity by FUL and miR172

Previous work showed that FUL binds to regulatory regions in the proximity of AP2-LIKE genes (AP2, TOE1, SNZ, and TOE3) and that these genes are expressed at higher levels in ful mutant inflorescences [47]. Whether these interactions were relevant during floral transition was previously unclear. We tested genetic interactions between ful and mir172 mutants under both LD and SD conditions (Figs 6 and 7). Under LDs, ful mir172abcd mutant plants flowered much later than either mir172abcd or ful plants. Furthermore, ful mir172abcd flowers exhibited a severe loss of determinacy, which did not occur in either parental line. Similarly, the longevity of ful mir172abcd plants was dramatically extended, with LD-grown plants continuing to produce flowers 4 months after germinating. This longevity is consistent with reports that the AP2-miR172 module is involved in global proliferative arrest [47]; however, it should be noted that the fertility of ful mir172abcd plants was extremely low. These phenotypes are closely related to those of plants in which AP2-LIKE genes are ectopically expressed [7,8,24,27,47]. Taken together, this genetic analysis indicates that FUL and miR172 control AP2-LIKE gene activity at both the transcriptional and posttranscriptional levels to regulate the floral transition, although this remains to be confirmed by examining expression of AP2 and AP2-L genes in the ful mir172abd background. Furthermore, as described above, analyzing transcript levels of these genes in different backgrounds is complicated by feedback regulation.

Expression analyses, including in situ hybridizations, indicate that miR172 acts prior to FUL at the shoot apex [35]. Therefore, we propose that miR172 is initially required to reduce AP2-LIKE activity and destabilize the vegetative state at the shoot meristem (Fig 7C). Furthermore, as AP2 directly represses FUL transcription [21], which is further supported by our analysis (S12F Fig), the depletion of AP2-LIKE TFs by miR172 is proposed to allow FUL transcription to increase. FUL would then bind to the promoters of AP2-LIKE genes initiating their transcriptional repression and thus enhancing the reduction of AP2-LIKE activity mediated by miR172 (Fig 7C). Affinity purification of FUL from inflorescences followed by protein mass spectrometry identified several chromatin remodeling proteins as interacting with FUL [62]. Therefore, binding of FUL to AP2-LIKE genes could mediate long-term transcriptional repression by compaction of the chromatin. We propose that these parallel mechanisms of posttranscriptional repression of AP2-LIKE gene expression by miR172 and transcriptional repression of the same genes by FUL provide a rapid and robust means to deplete these potent negative regulators of floral transition from the induced shoot apex as flowering proceeds (Fig 7C).

Expression of FUL and MIR172 appears to be partially coordinated by SPL15 under both LDs and SDs, but its contribution is more important under SDs. When grown in SDs, FUL and miR172 are undetectable by RNA in situ hybridization in spl15 mutants and SPL15 binds to the promoter regions of FUL and MIR172B. However, given that mir172abd mutants flower much later than spl15-1 with FUL expression persisting in their SAMs (S12F Fig) and inactivation of FUL or MIR172A/B can strongly enhance the phenotype of spl15-1 (Fig 7A), miR172 and FUL activity must still occur in spl15-1. However, analysis of transgenic plants ectopically expressing a miR156-resistant version of SPL15 demonstrated that SPL15 can promote the transcription of MIR172A, MIR172B, MIR172C, and MIR172D (Fig 6E–6J). Furthermore, genetic analysis demonstrated that FUL and miR172 activity are partially required for rSPL15 activity, while combinations of ful and mir172 mutants can partially phenocopy the spl15 mutant (Figs 6K and 6L and 7A and 7B). These data support the notion that SPL15 acts through both FUL and MIR172 to promote flowering, but that SPL15 also acts independently of these factors (Fig 7C). Similarly, other unidentified regulators must promote FUL and MIR172 expression in parallel with SPL15. Thus, SPL15 mediates the transition from the stable vegetative state to the reproductive state by activating at least 2 repressors of AP2-LIKE TFs that act partially redundantly at different levels of regulation (Fig 7C). This is a form of feedforward circuit that has been described in other systems to mediate robust and stable repression of miRNA targets [60,63] and here likely plays a role in stabilizing the floral state.

Plant growth and strains used

Plants were grown on soil in LD (16 h light/8 h dark) or SD (8 h light/16 h dark) conditions at 18 to 22°C with a light intensity between 150 and 160 μmol m−2 s−1. All genotypes were in the A. thaliana (L.) ecotype Columbia-0 (Col-0) background. Previously published A. thaliana strains include ap2-12 [21], ful-2 [50], 35Spro:MIM172 [5], spl15-1 [38], and Venus-rSPL15 [35]. The following strains were generated in this study and are described below: MIR172A-NVG, MIR172B-NVG, MIR172C-NVG, MIR172D-NVG, MIR172E-NVG, mir172a-2, mir172b-3, mir172c-1, mir172d-2, mir172d-3, mir172e-1, mir172a-2 b-3, mir172a-2 b-3 c-1, mir172a-2 b-3 c-1 d-3, mir172a-2 b-3 c-1 d-3 e-1, mir172a-2 b-3 d-2, mir172a-2 b-3 d-2 ap2-12, spl15-1 ful-2, spl15-1 mir172a-2 b-3, ful-2 mir172a-2, ful-2 mir172b-3, ful-2 mir172a-2 b-3, Venus-rSPL15 ful-2 mir172a-2 b-3, Venus-rSPL15 ful-2, Venus-rSPL15 mir172a-2 b-3, AP2_genomic-Venus, ful-2 mir172a-2 b-3 c-1 d-3; first generation plants harboring UBQ10pro:CTRL, UBQ10pro:MIR172A, UBQ10pro:mir172a-1, UBQ10pro:mir172a-2, UBQ10pro:MIR172B, UBQ10pro:mir172b-1, UBQ10pro:mir172b-3, UBQ10pro:MIR172D, UBQ10pro:mir172d-2, UBQ10pro:mir172d-3, UBQ10pro:MIR172E and UBQ10pro:mir172e-1.

Cloning MIR172-NVG reporter constructs

Polymerase Incomplete Primer Extension (PIPE) [64] was used to generate the NVG-stb205 and MIR172(A-E)-NVG-stb205 plasmids. The NVG-stb205 plasmid, which is a variant of pDONR201, was generated with primers oDM-462-465 (S2 Table). To generate the MIR172(A-E)-NVG-stb205 plasmids, initially, the entire intergenic region containing the MIR172 coding region of each gene was placed in stb205 with primers oDM-11-20 (S2 Table). The NVG coding region was used to replace the miRNA hairpin coding sequence in each MIR172 gene using primers oDM-31-50 (S2 Table). Either Phusion (New England Biolabs, United States of America) or PrimeSTAR GXL (TaKaRa Bio, Japan) high-fidelity polymerases were used for DNA amplification. The PCR products derived from plasmid templates were treated with DpnI (New England Biolabs) overnight at 37°C to remove the template DNA. After purification of each PCR product with the NucleoSpin PCR clean-up kit (Macherey Nagel, Germany), a molar ratio of 1:10 plasmid:insert PCR products were mixed and 100-μL chemically competent Dh5α Escherichia coli were transformed using the heat shock method. Transformed bacteria were incubated with 500-μL LB for 1 h at 37°C in a shaker. The entire liquid culture was then centrifuged for 5 min at 5 krpm, and 500 μL of the supernatant was removed. The other 100 μL of supernatant was used to resuspend the bacterial pellet, which was plated on LB-agar plates supplemented with 50 μg/mL kanamycin and incubated at 37°C overnight. Colonies were tested by PCR using primers in S2 Table, and positive colonies were used to inoculate 5-mL liquid LB culture, which were supplemented with 50 μg/mL kanamycin and were grown overnight at 37°C. Plasmids were extracted using the NucleoSpin Plasmid extraction kit (Macherey Nagel, Germany), and the success of the cloning was initially determined by DNA restriction enzyme analysis. Plasmids with the correct restriction patterns were subjected to Sanger sequencing (Eurofins Genomics, Germany) to verify that the correct sequences were present in the plasmids. MIR172(A-E)-NVG sequences were then transferred to the p*GW binary vector [65] using the Gateway LR clonase II enzyme mix (Invitrogen, USA), according to the manufacturer’s protocol. After transformation protocols as described above, a 100-μL culture was plated on LB-agar plates supplemented with 100 μg/mL spectinomycin. Colonies were tested by PCR using primers in S2 Table. The success of the cloning was determined as described above although Sanger sequencing was not repeated. Plasmids were transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and 500 μL of LB was added and incubated at 28°C for 3 to 4 h. Moreover, 100 μL of the culture was plated on LB-agar plates supplemented with 100 μg/mL spectinomycin, 100 μg/mL rifampicin, and 25 μg/mL gentamicin and incubated at 28°C. Positive colonies were determined by colony PCR using primers in S2 Table, and cultures were grown in LB medium supplemented with 100 μg/mL spectinomycin, 25 μg/mL gentamicin, and 100 μg/mL carbenicillin.

Cloning overexpression of wild-type and mutant MIR172 alleles

The A. thaliana UBIQUITIN10 promoter and the octopine synthase terminator sequences (from pBJ36 [66]) were introduced to the 5′ and 3′ sides of the multiple cloning site of pGREEN-0229 [67], respectively, using restriction enzyme based cloning to generate pGREEN-0229-UBQ10pro-3′OCS (S2 Table). The WT and mutant alleles for MIR172 were amplified with the Phusion (New England Biolabs) high-fidelity polymerase and primers that contained XhoI and NotI sites (S2 Table). PCR products and pGREEN-0229-UBQ10-3′OCS were incubated with XhoI and NotI (New England Biolabs) overnight at 37°C. After gel extraction and purification of the digested products with the NucleoSpin PCR clean-up kit (Macherey Nagel, Germany), they were incubated at 4°C overnight with T4 DNA ligase (New England Biolabs). The ligation reaction was then transformed into Dh5α using the heat shock method. Transformed bacteria were incubated with 500 μL of LB for 1 h at 37°C in a shaker. Moreover, 100 μL of the supernatant was plated on LB-agar plates supplemented with 50 μg/mL kanamycin and incubated at 37°C overnight. Success of the cloning, transformation of A. tumefaciens strain GV3101 that contained the pSOUP helper plasmid, and culturing of positive colonies was performed as described above with the exception that 10 μg/mL of tetracycline was also added to the LB/LB-agar media.

Single guide RNA cloning, T7E1 assays, and CRISPR genotyping

Single guide RNA (SgRNA) cloning was performed according to the methods in [52,53], and the appropriate primers are listed in S2 Table. Pure genomic DNA was extracted from the cauline leaves of T1 plants [68]. The region in which the Cas-9 protein was targeted was flanked by 2 sets of nested primers (S2 Table). Amplified products were reannealed by denaturing them at high temperature and slowly decreasing the temperature [53]. Moreover, 1 μL of the T7E1 enzyme (New England Biolabs) was then introduced to 10 μL of the PCR reaction supplemented with sterile distilled H₂O and the appropriate buffer (NEB), and samples were incubated at 37°C for 20 min in a thermocycler. Incubated products were then run on an EtBr-supplemented 3% agarose gel and visualized under an ultraviolet lamp (S4 Fig). Seeds from plants that were positive in the T7E1 assay were then planted, and plants with stable mutations were identified through a combination of T7E1 assays, PCR amplification, and Sanger sequencing. Plants lacking the Cas-9 insertion were identified using a leaf-based phosphinothricin (PPT) resistance assay (S4N Fig) and genotyping (S2 Table).

Cloning of AP2_genomic-Venus reporter construct

PIPE [64] was used to generate the plasmids csAP2-pDONR201, pAP2-AP2-pDONR201, pAP2-AP2-Venus-pDONR201, and pAP2-AP2-Venus-pEarlyGate301. All PCR amplifications were done with Phusion Enzyme (New England Biolabs) following the manufacturer’s recommendations. Amplification of the AP2 coding sequence was performed with Q236+Q272 and was cloned into pDONR201 by BP reaction to generate csAP2-pDONR201. Q273+fa20r were used to linearize csAP2-pDONR201, and Q274+Q275 were used to amplify the AP2 promoter to generate pAP2-AP2-Venus-pDONR201. The Venus coding region was amplified using Q058+Q359, and Q357+Q358 were used to linearize pAP2-AP2-Venus-pDONR201 to generate pAP2-AP2-Venus-pDONR201. The pAP2-AP2-Venus sequence was then transferred to the pEarlyGate binary vector [65] using the Gateway LR clonase II enzyme mix (Invitrogen), according to the manufacturer’s protocol. Transformation of bacteria was performed as described above.

Plant transformation and selection

The floral dip method [69] was used to transform A. thaliana. A solution containing the appropriate A. tumefaciens strain, 10% w/v sucrose, and 0.025% v/v Silwett L-77 was used to dip the flowers of A. thaliana. Plants were then incubated in the dark at room temperature overnight before being placed in LD-grown conditions. For selection of plants transformed with a bar selection cassette, seeds were germinated on soil, and seedlings were sprayed with 200 μg/ml ammonium-glufosinate. For selection of plants transformed with a hygromycin-resistance selection cassette, sterilized seeds were plated on MS agar supplemented with 12 μg/ml hygromycin, covered, and placed in a growth chamber for 3 d. After 3 d, the cover was removed, and seedlings were grown until a noticeable difference in hypocotyl length was observed. Seedlings with longer hypocotyls were presumed to be resistant to hygromycin. Putative transformants were genotyped using appropriate primers for confirmation.

Phosphinothricin (PPT) resistance assay

Personal communications with several researchers from independent groups highlighted the problem with segregating the Cas-9 containing T-DNAs from the mutation of interest, as they often appeared to be genetically linked. In order to identify plants lacking the Cas-9 containing T-DNAs without needing to genotype hundreds of plants or propagate seeds from hundreds of plants, we used a plate based PPT-resistance leaf assay. MS-agar plates (without sucrose) supplemented with 25 μg/mL PPT were poured. A leaf (preferably a young leaf but part of an old leaf is also suitable) was placed on the MS-agar and pressed down until it stuck to the agar. Plates were then sealed with medical tape and placed in a growth cabinet for 2 to 3 days. Leaves derived from PPT-resistant plants were dark green, whereas leaves from PPT-susceptible plants were light green (S4N Fig). Follow-up genotyping by PCR confirmed the absence or presence of the Cas-9 T-DNA (S2 Table). This assay was not performed in sterile conditions, and fungal growth was kept to a minimum by using sucrose-free MS-agar plates and limiting the time the MS-agar plates were open.

ClearSee and confocal imaging

The ClearSee reagent (10% (w/v) xylitol, 15% (w/v) sodium deoxycholate, and 25% (w/v) urea) was prepared as previously described [70]. Tissue was dissected at approximately zeitgeber time (ZT) 6 and placed in 4°C 4% paraformaldehyde prepared in PBS (pH 7), which was resting on ice. Tissue was then subjected to a vacuum of approximately 700 mbar at room temperature for 1 h. The vacuum was then released, and samples were incubated for a further 1 h. Samples were washed with 1x PBS 3 times and then incubated with ClearSee in the dark for up to 14 d. The shoot meristems were visualized with a LSM780 (Zeiss, Germany) laser scanning confocal microscope, and the settings were optimized for the visualization of the Venus fluorescent protein (laser wavelength, 514 nm; detection wavelength, 517 to 569 nm) and the SCRI Renaissance 2200 dye (laser wavelength, 405 nm; detection wavelength, 410 to 503 nm), which stains plant cell walls [71]. The same settings were used for each sample; however, the gain for visualizing the plant cell walls by Renaissance staining was modified for presentation purposes. This does not change the interpretation of the results, and original images are available upon request. The Renaissance dye was added to the samples 12 to 24 h before imaging. For Fig 5A, the samples in a Col-0 and mir172abd background are unpaired. The Col-0 background samples were imaged with a lower 514-nm gain. A paired Col-0 background sample was performed, but the signal was dimmer probably because of the quenching effects of ClearSee; however, the data were similar.

RNA extraction and RT-qPCR

Plant tissue was harvested at the indicated times on liquid nitrogen and immediately frozen at −80°C. Samples were processed with the RNeasy Plant Mini Kit (Qiagen, the Netherlands) according to the manufacturer’s protocol. RNA was quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, USA), and up to 5 μg of RNA was incubated with DNase I (TURBO DNA-free kit, Invitrogen) to remove genomic DNA according to the manufacturer’s protocol. After quantification, 1 μg of DNA-free RNA was used in a reverse transcription reaction (Superscript IV first-strand synthesis system, Invitrogen) and diluted 1:5 with pure and sterile H₂O. Moreover, 1 μL of the diluted reverse transcription reaction was used in a PCR reaction with iQ™ SYBR green supermix (Bio-Rad, USA). Relative transcript abundance was determined with a Roche LightCycler 480 system. Measurements were taken for 3 independent biological triplicates, and the average transcript abundance for each biological sample was determined by performing 2 to 3 technical repeats. LightCycler melting curves were obtained for the reactions, revealing single peak melting curves for most amplification products. The amplification data were analyzed using the second derivative maximum method, and resulting Cp values were converted into relative expression values using the comparative cycle threshold method [72]. One reference gene (REF1 (PROTEIN PHOSPHATASE 2A (PP2A)) was used to normalize the data.

DNA extraction and genotyping

Pure genomic DNA was extracted for T7E1 assays according to [68], for next generation sequencing with the DNeasy plant mini kit (Qiagen) according to manufacturer’s instructions, and for genotyping according to [73]. Platinum Taq DNA polymerase (Invitrogen) was used for all PCR assays except for cloning DNA fragments.

Statistical analysis

We have implemented some recommendations from The American Statistician [74]. Specifically, we avoid the term “statistically significant,” and we report p-values as continuous where possible. We have supplied a Supporting information table in which all p-values are reported in addition to a description of the dataset (S1 Table). Although grouping data based on statistical thresholds is not recommended [74], we have provided a grouping threshold in Table 1 and S1 Table as we believe that it can be a helpful guide, but we do not interpret our results on the basis of these groupings.

Data were analyzed using the R studio software [75]. The distribution and variance of the data were analyzed initially by visualization and the base functions shapiro.test, and bartlett.test and/or fligner.test, respectively. Data were analyzed using 1-way analysis of variance (ANOVA) if data were deemed to be normally distributed (according to a combination of statistical analysis and data visualization) and if the variance of the data were equal (p > 0.01 for at least one of bartlett.test or fligner.test). If these assumptions were violated, data were transformed to meet these assumptions. The Kruskal–Wallis test was used (kruskal.test) if the data were not normally distributed; however, p > 0.01 was required for at least one of bartlett.test or fligner.test. If p < 0.01 for bartlett.test and fligner.test, and data transformation did not off-set the problem, the Brown–Forsythe (bf.test from the package onewaytests) test was implemented [76].

Post hoc tests were employed if a significant result was obtained from the above tests (p < 0.01). The base function pairwise.t.test was used following an ANOVA test, with analysis being paired or unpaired depending on the experimental design. Dunn test (dunn.test [77]) was used following the Kruskal–Wallis test. Games Howell test was used following the Brown–Forsythe test from the package userfriendlyscience [78]. Adjustments for multiple corrections were performed in each case using the Benjamini–Hochberg method [79].

Tests for genetic synergism were done by subtracting observed values from the mean of the reference Col-0. Then means and variances were added to simulate the expected additive effect of the 2 alleles. This was compared with the observed differences between the higher-order mutant and Col-0 using an unpaired 2-tailed Welch t test.

For the box plots, the box encapsulates the 25th to 75th percentile, and the error bars encapsulate the 10th and 90th percentiles. The horizontal line running through the box indicates the median, and each point represents an individual plant. Bars represent the mean value, and the error bars represent the standard error of the mean (SEM). Graphs were modified for style using Adobe Illustrator.

GUS staining

GUS staining was performed according to [80] with some modifications. Briefly, samples were harvested at approximately ZT6 in ice-cold 90% acetone and vacuum infiltrated at approximately 700 mbar for 10 min at room temperature. The vacuum was then released, and samples were incubated for a further 20 min. The acetone was removed and replaced with cold staining buffer (50 mM PO₄, 0.2% Triton X-100, 2 mM ferro-ferricyanide) without X- glucuronidase (X-gluc) and vacuum infiltrated at approximately 700 mbar on ice for 10 min. Cold staining solution without X-gluc was removed, and staining solution with 2 mM X-gluc was added, and samples were vacuum infiltrated on ice for up to 20 min with the vacuum being released and reapplied 2 to 3 times. Samples were then incubated at 37°C for 2 h or overnight. After this incubation, samples were submerged in 70% ethanol, which was refreshed until the chlorophyll was removed. Images were taken with a Zeiss SMZ-25 (Zeiss) stereomicroscope or a digital SLR camera.

RNA in situ hybridization

Plants were grown in LD conditions for the specified time. In situ hybridization was performed as previously described [59]. The osa-miR172a miRCURY LNA miRNA detection probe (Qiagen, Cat No. YD00618724) was used to detect miR172 RNA. A probe for AP2 was synthesized as described in [31].

Whole genome sequencing and off-target analysis

Library preparations of genomic DNA derived from quintuple mir172abcde mutant plants were performed using the NEBNext Ultra II FS DNA library preparation kit for Illumina. Whole genome sequencing (20X coverage) was performed with an Illumina HiSeq3000 with single-end 150 bp reads. Potential off-targets were identified by comparing the sgRNA sequences with the A. thaliana genome, allowing for up to 5 mismatches using the dedicated off-target search program CAS-OFFinder [81]. In order to detect mutations, the raw single-end reads were processed by removing the adapter sequences using cutadapt [82] and then trimming low-quality ends using Trimmomatic [83]. The cleaned reads were mapped to the A. thaliana TAIR10 genome using BWA [84] with default settings. BWA outputs both primary, and if found, secondary alignments. In this study, we only retained primary alignments with a mapping quality of at least 30. Samtools mpileup [85] (default settings) was used in conjunction with bcftools [86] for initial SNP calling. SNPs were filtered by minimum quality of 25, minimum depth of 5. Additionally, all regions of the genome that were devoid of any mapped read were labeled as potential larger deletions. Finally, we searched for overlap between the predicted off-targets, the SNPs, and larger deletions.

RNA secondary structure analysis

Sequences from 24 nucleotides downstream and upstream of the miR172/miR172* duplex coding sequences of WT and mutant MIR172 alleles were processed by mFold version 2.3 [87] with standard settings except that the temperature was set at 22°C. The predicted structure with the lowest minimum free energy is presented.

RNA extraction and RNA sequencing transcript profiling

The shoot apex of Col-0 and mir172abd were dissected under a stereo microscope at 10, 12, 14, and 17 days in LD conditions and 3, 4, 5, and 6 weeks in SD conditions in 3 independent biological replicates. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, USA) and subjected to DNase treatment using the TURBO DNase (Invitrogen). Poly(A) RNA enrichment, library preparation, and sequencing were carried out at the MPIPZ Genome Center, Cologne, Germany using the following conditions: The RNAs were processed by poly-A enrichment followed by application of basic components of “NEBNext Ultra II Directional RNA Library Prep Kit for Illumina” with a homebrew barcoding regime. Sequencing was performed on a HiSeq3000 sequencer by sequencing-by- synthesis with 1 × 150 bp single-read length. Sequence reads were preprocessed to remove any residual adaptors with CutAdapt, and the low-quality bases (Q<15) were trimmed from the ends with Trimmomatic [82,83]. Only reads with a minimum length of 50 nucleotides were kept. Salmon was used to quantify the abundance of transcripts from the Arabidopsis reference genome Reference Transcript Dataset for Arabidopsis (including guanine/cytosine bias, unstranded samples) [88,89]. Fragments Per Kilobase of transcript per Million (FPKM) values and corrected p-values were obtained using DESeq2 by comparing Col-0 to mir172abd in each time point using standard settings. Data from this RNA-seq transcript profiling are deposited in the BioProject database (https://www.ncbi.nlm.nih.gov/bioproject) under the accession number PRJNA669254.

Gene accession numbers

Annotations and sequence data of the genes studied in this article can be found at The Arabidopsis Information Resource (TAIR) under the following accession numbers: MIR172A (At2g28056), MIR172B (At5g04275), MIR172C (At3g11435), MIR172D (At3g55512), MIR172E (At5g59505), AP2 (At4g39620), TOE1 (At2g28550), TOE2 (At5g60120), TOE3 (At5g67180), SNZ (At2g39250), SMZ (At3g54990), FUL (At5g60910), SPL15 (At3g57920), FT (At1g65480), and AP1 (At1g69120).