Abstract
In Arabidopsis, loss of the carboxypeptidase, ALTERED MERISTEM PROGRAM1 (AMP1), produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 may promote SPL activity. However, we found that the amp1 phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although evidence from a previous study suggests that AMP1 promotes miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target, SPL9, or the miR159 target, MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway and that it is not universally required for miRNA-mediated translational repression.
Summary statement We show that loss of the carboxypeptidase, AMP1, does not interfere with the function of miR156 or miR159, suggesting that AMP1 is not universally required for miRNA-mediated translational repression in Arabidopsis.
INTRODUCTION
Plant life-histories are underpinned by a series of developmental transitions, the correct timing of which are crucial to plant survival and reproductive success (Huijser and Schmid, 2011). Vegetative phase change describes the switch between the juvenile and adult stages of vegetative growth. Depending on the species, this transition can lead to shifts in a wide variety traits (Poethig, 2013). In the model plant, Arabidopsis thaliana, the juvenile vegetative phase is associated with small, round leaves that lack both trichomes on the abaxial leaf surface and serrations, whereas the adult phase is characterized by larger, elongated and serrated leaves that produce abaxial trichomes.
The core genetic network that controls the timing of vegetative phase change has been well described. The microRNA miR156, and its sister miR157, function as master regulators of the juvenile phase. A temporal decline in miR156/miR157 during shoot development leads to an increase in expression of their target genes—SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors—which promote the adult phase (Wu and Poethig, 2006; Wu et al., 2009). This temporal mechanism is widely conserved and regulates shoot identity in diverse plant lineages (Chuck et al., 2007; Leichty and Poethig, 2019; Riese et al., 2008; Wang et al., 2011). SPL genes are known to promote the expression of miR172, which initiates adult development through repression of its targets in the APETALA2-LIKE (AP2-LIKE) gene family. Vegetative phase change is thus promoted by inverse gradients of expression of two miRNAs, miR156 and miR172 (Wu et al., 2009).
ALTERED MERISTEM PROGRAM1 (AMP1), which encodes a putative carboxypeptidase (Helliwell et al., 2001), was identified in a genetic screen for phase change mutations over 20 years ago (Conway and Poethig, 1997), but the basis for its effect on this process is still unknown. Mutations in AMP1 produce a large number of small, round leaves that lack abaxial trichomes (juvenile leaves) and have a higher rate of leaf initiation (Telfer et al., 1997). An initial study suggested that this phenotype was not associated with a change in the timing of vegetative phase change, leading to the conclusion that the timing of vegetative phase change is regulated independently of leaf number (Telfer et al., 1997). However, this result conflicts with more recent studies showing that pre-existing leaves promote vegetative phase change (Yang et al., 2011; Yang et al., 2013; Yu et al., 2013). The phenotype of amp1 is also surprising given the evidence that AMP1 is required for miRNA-mediated translational repression (Li et al., 2013). miR156 promotes juvenile development by translationally repressing its targets (He et al., 2018). If AMP1 is required for miRNA-mediated translational repression, amp1 mutants would therefore be expected to have to a reduced number of juvenile leaves due to elevated SPL gene expression, which is the exact opposite of the amp1 phenotype.
To resolve these issues, we investigated the interaction between AMP1 and the miR156-SPL module. Our results indicate that AMP1 promotes adult leaf traits in parallel to, or downstream of, the miR156-SPL module. We also found no evidence that AMP1 is required for translational repression by either miR156 or miR159. This latter result suggests that the mechanism by which miRNAs repress translation in plants is different for different transcripts.
RESULTS AND DISCUSSION
Elevated SPL activity has a modest effect on the amp1 phenotype
amp1-1 (hereafter, amp1) mutants resemble plants with reduced SPL gene expression in having an increased rate of leaf initiation, an increased number of rosette leaves, an enlarged shoot apical meristem, and small, round rosette leaves that lack abaxial trichomes (Fig. 1A-F) (Chaudhury et al., 1993; Huang et al., 2015; Telfer et al., 1997; Yang et al., 2018). To determine if this phenotype is attributable to a reduction in SPL activity, we introduced 35S::MIM156 — which de-represses SPL gene expression (Franco-Zorrilla et al., 2007) — into amp1. 35S::MIM156 plants have a relatively slow rate of leaf initiation, have enlarged and somewhat elongated rosette leaves, produce abaxial trichomes unusually early in shoot development, and have a relatively small SAM (Fig. 1A-F). amp1; 35S::MIM156 plants had a vegetative phenotype intermediate between that of the two parental genotypes, but which was more similar to amp1 than to 35S::MIM156. The rosette leaves of amp1; 35S::MIM156 were approximately the same size as amp1 leaves, but were similar in shape to 35S::MIM156 (Fig. 1A, B). amp1 plants rarely produced rosette leaves with abaxial trichomes (although abaxial trichome production on cauline leaves was unaffected (Fig. S1)), whereas about 25% of amp1; 35S::MIM156 produced rosette leaves with abaxial trichomes late in shoot development. In contrast, all 35S::MIM156 plants produced rosette leaves with abaxial trichomes by plastochron 3 (Fig. 1C). Similarly, the rate of leaf initiation in amp1; 35S::MIM156 was intermediate between that of amp1 and 35S::MIM156, but was closer to that of amp1 than 35S::MIM156 (Fig. 1D). The number of rosette leaves in amp1; 35S::MIM156 was also intermediate between these two genotypes, but was more similar to amp1 than 35S::MIM156 (Fig. 1E). Finally, the SAM of amp1; 35S::MIM156 was more similar in size to amp1 than to 35S::MIM156 (Fig. 1F). These results suggest that the phenotype of amp1 is not a consequence of repressed SPL activity, implying that AMP1 acts either downstream or in parallel to the miR156/SPL module. This conclusion is consistent with the observation that vegetative development (Fig. 1C) and flowering time (Fig. 1G) are dissociated in amp1.
The phenotype of amp1 is not attributable to a change in miR156/miR157 or SPL gene expression
To explore the relationship between AMP1 and the miR156/SPL module in more detail, we examined the effect of amp1 on the abundance of the miR156 and SPL transcripts. qRT-PCR analysis of the shoot apices of plants grown in short days (SD) showed that amp1 had no significant effect on the level of miR156 or miR157 (Fig. 2A), or the transcripts of three direct targets of these miRNAs: SPL3, SPL9 and SPL13 (Fig. 2B). To test whether amp1 affects SPL expression independent of miR156/miR157, we measured the transcripts of these genes in 35S::MIM156 and amp1; 35S:MIM156 plants. As expected (He et al., 2018), all three SPL transcripts were significantly elevated in 35S::MIM156. All three transcripts were elevated to a much smaller extent in amp1; 35S:MIM156 (Fig. 2B). Together, these results suggest that AMP1 may promote SPL expression, but only in the absence of miR156/miR157.
We then examined the expression of these genes in successive rosette leaf primordia (LP) of plants grown in SD. Because amp1 initiates leaves more rapidly than WT, LP were grouped according to the time of harvest rather than position on the shoot. Both the level and rate of decline of miR156 were almost identical in WT and amp1 (Fig. 2C). miR157 was elevated in all LP, but declined at approximately the same rate as in WT plants. SPL9 and SPL13 transcripts were also elevated in the LP of amp1 relative to WT (Fig. 2D), but these differences were relatively modest (two-fold or less) and not statistically significant. Furthermore, the elevated expression of SPL9 and SPL13 is inconsistent with the elevated level of miR157 and with the juvenilized phenotype of amp1. Taken together, these data suggest that the vegetative phenotype of amp1 is not caused by increased expression of miR156/miR157 or decreased expression of SPL genes. It is possible that AMP1 regulates SPL expression independently of miR156 (Fig. 2B). However, the observation that amp1 does not have a significant effect on SPL9 and SPL13 expression at 20 DAP (Fig. 2D), when the levels of miR156 and miR157 are very low (Fig. 2C), suggests that this is unlikely.
If AMP1 does not regulate miR156 or SPL gene expression, perhaps it regulates shared downstream targets. Consistent with this hypothesis, expression of the closely-related AP2-like transcription factors TOE1 and TOE2 (which are targets of the SPL-regulated miRNA, miR172) was consistently elevated in amp1 (Fig. 2E). This effect is not attributable to a change in the level of miR172, however, as the abundance of this miRNA was not reduced in amp1 (Fig. 2C). TOE1 blocks the production of trichomes on the abaxial side of the leaf by working in association with the abaxial specification gene KANAD1 (KAN1) to repress the transcription of GLABRA1 (GL1) (Wang et al., 2019; Xu et al., 2019). In WT plants, GL1 expression increased dramatically between 13-14 DAP and 20 DAP, consistent with the increase in trichome production over this period. GL1 displayed a similar temporal pattern in amp1, but was almost completely suppressed in the earliest LP and was considerably lower than WT in LP harvested at DAP (Fig. 2F). In contrast, the expression of TRANSPARENT TESTA GLABRA1 (TTG1) — which promotes trichome initiation via a distinct protein complex to GL1 (Pesch et al., 2015) — was not reduced in amp1 (Fig. 2G). These results suggest that AMP1 promotes abaxial trichome formation via GL1, not TTG1, and that it acts as a general activator of GL1 expression, rather than a temporal regulator. They also support the conclusion that AMP1 regulates abaxial trichome production downstream of miR156/SPL.
The timing of vegetative phase change is regulated independently of leaf initiation in amp1
The juvenilized phenotype of amp1 was originally attributed to the increased rate of leaf initiation in this mutant (Telfer et al., 1997). However, this interpretation is inconsistent with more recent studies showing that pre-existing leaves promote the transition to the adult vegetative phase by repressing miR156 (Yang et al., 2011; Yang et al., 2013; Yu et al., 2013). To determine the basis of this discrepancy, we characterized the effect of CLAVATA3 (CLV3) and CLV1 mutations on vegetative phase change. We chose these mutations because they resemble amp1 in having an enlarged SAM and an accelerated rate of leaf initiation (Clark et al., 1995; Leyser and Furner, 1992).
Like amp1 (Telfer et al., 1997), clv3 and clv1 produced smaller, rounder rosette leaves, and more leaves without abaxial trichomes (Fig. 3A - C). This increase in the number of juvenile-like leaves was not associated with a delay in the juvenile-to-adult transition, however. Instead, clv3 mutants produced leaves with abaxial trichomes one day earlier than WT plants (Fig. 3D). To determine if the phenotype of clv1 and clv3 is dependent on miR156, we introduced the miR156 sponge, 35S::MIM156, into these mutants. This transgene was epistatic to clv1 and clv3 with respect to their effect on leaf shape (Fig. 3A, B) and abaxial trichome production (Fig. 3C), suggesting that their effect on these traits requires miR156.
We then examined the effect of clv3 and clv1 on the expression of miR156 and its targets, SPL9 and SPL13, in shoot apices (Fig. 3E) and LP (Fig. 3F). qRT-PCR revealed that clv1 and clv3 have slightly reduced levels of miR156, although this difference was only statistically significant in clv3. Consistent with the decreased amount of miR156, SPL9 and SPL13 transcripts were slightly elevated in both the mutants, although again this difference was only statistically significant in a few cases. If these relatively small differences in miR156 and SPL gene expression are functionally significant, they would be expected to promote the appearance of adult traits, not repress the expression of these traits as is the case in clv1 and clv3. To explore this inconsistency, we examined the effect of clv3 on the expression of a miR156-sensitive and a miR156-resistant version of the SPL9::SPL9-GUS reporter (Xu et al., 2016). There was no obvious difference in the expression of these reporters in the presence or absence of clv3 (Fig. 3G), supporting the conclusion that the effect of clv3 on leaf identity is not attributable to a change in the level of miR156 or its targets.
Instead, the effect of clv3 and clv1 on leaf identity is primarily attributable to their effect on the rate of leaf initiation. Specifically, clv3 and clv1 appear to increase the number of juvenile leaves by accelerating the rate of leaf production during the period when miR156 levels are high. This conclusion is supported by the observation that 35S::MIM156 is epistatic to these mutations with respect to their effect on leaf identity (Fig. 3A, B); i.e. miR156 is required for their leaf identity phenotypes. Consistent with the evidence that leaves promote the juvenile-to-adult transition by repressing miR156 (Yang et al., 2011; Yang et al., 2013; Yu et al., 2013), clv3 and clv1 have slightly reduced levels of miR156 and slightly elevated levels of SPL9 and SPL13 (Fig. 3E, F). However, this relatively small effect is apparently insufficient to interfere with the function of these genes in specifying juvenile leaf identity.
The increased number of juvenile leaves in amp1 is also partly attributable to its higher rate of leaf initiation (Telfer et al., 1997). However, amp1 differs from clv3 and clv1 in having a much more significant effect on leaf identity: amp1 rarely produces abaxial trichomes on rosette leaves, whereas clv3 and clv1 routinely do so. In addition, the phenotype of amp1 is less sensitive to a reduction in miR156 than the phenotype of clv3 and clv1; in general, amp1, 35S::MIM156 plants more closely resembled amp1 than 35S::MIM156 (Fig. 1A-E). This observation, and the effect of amp1 on the expression of genes involved in abaxial trichome production (Fig. 2E, F), suggest that AMP1 operates independently of miR156 to regulate genes involved in leaf identity. A direct effect of AMP1 on leaf identity genes would explain why amp1 has a more severe vegetative phenotype than clv3 and clv1, and why the phenotype of amp1 is relatively insensitive to changes in the level of miR156.
AMP1 is not universally required for translational repression
Given the role of AMP1 in translational repression (Li et al., 2013), it is possible that the abundance of SPL transcripts in amp1 (Fig. 2B, D) does not accurately reflect their biological activity. To determine whether AMP1 is required for the post-transcriptional regulation of SPL genes, we first measured the amount of SPL9 and SPL13 transcript cleavage in WT and amp1 plants. Consistent with a previous study on miR156-mediated cleavage (He et al., 2018), the rate of transcript cleavage for both SPL9 and SPL13 declined during vegetative development in WT plants (Fig. 4A). This happened at a slower rate in amp1, presumably in part due to the higher level of miR156 in the amp1 13-14 DAP sample compared to WT (Fig. 2C) and the threshold-dependence of miR156 activity (He et al., 2018). However, later in development, transcript cleavage in amp1 was similar to WT (Fig. 4A). This demonstrates that miR156 is functional in amp1 and confirms the observation that AMP1 is not required for transcriptional cleavage (Li et al., 2013).
Although miR156 induces transcript cleavage, it represses the expression of its targets primarily by promoting translational repression (He et al., 2018). To examine the effect of amp1 on this process, we crossed miR156-sensitive (sSPL9) and miR156-resistant (rSPL9) GUS-reporter constructs of SPL9 into amp1. There was no obvious difference in the staining intensity of these reporter proteins in WT and amp1 (Fig. 4B). To confirm this impression, we measured the staining intensity of the sSPL9-GUS reporter spectrophotometrically in leaf primordia of WT and amp1 harvested at a stage when transcript cleavage was nearly equivalent in these genotypes (20 DAP (Fig. 4A)). There was no significant difference in sSPL9 protein levels in these genotypes (Fig. 4C, D). These results indicate that amp1 has no effect on the activity of miR156, implying that translational repression of SPL9 occurs normally in amp1. To determine if miR156 is uniquely insensitive to amp1, we examined the effect of amp1 on the expression of MYB33, a transcription factor that also regulates shoot identity (Guo et al., 2017) and is translationally repressed by miR159 (Li et al., 2014). miR159-sensitive and miR159-resistant versions of MYB33-GUS (Millar and Gubler, 2005) were crossed into amp1, and WT and amp1 plants were stained for GUS activity one week after germination, and at flowering. MYB33-GUS was repressed in a miR159-dependent fashion in leaves and floral organs of WT plants, and amp1 had no obvious effect on this expression pattern (Fig. 4E, F). Because amp1 had no effect on the expression of sMYB33-GUS, it is reasonable to assume that miR159-dependent translational repression occurs normally in this mutant. We conclude from these results that AMP1 is not universally required for the translational repression of miRNA-targets.
Whether or not AMP1 functions in translational repression may be a result of the sub-cellular localization of the process. AMP1 has been shown to colocalize with the key silencing component ARGONAUTE1 (AGO1) on the endoplasmic reticulum (ER) (Li et al., 2013). However, AGO1 also localizes to processing bodies (p-bodies), cytoplasmic foci of mRNA-ribonucleoprotein complexes that facilitate the sequestration of mRNAs for translational silencing (reviewed in Chantarachot and Bailey-Serres, 2018). Loss of the p-body protein SUO leads to a reduction in the translational repression of the miR156-target SPL3 (Yang et al., 2012), suggesting that p-bodies are also important sites of miRNA-mediated translational repression. Taken together, these results are consistent with a model in which a) miRNA-mediated translational repression occurs in distinct sub-cellular compartments in a sequence-specific manner and b) unique sets of proteins contribute to this repression, depending on the compartment (e.g. AMP1 on the ER, SUO in p-bodies). Whether the translational repression of MYB33 by miR159 occurs in p-bodies remains to be demonstrated.
Support for this model comes from the finding that the microtubule severing-enzyme KATANIN 1 is also required for translation repression (Brodersen et al., 2008). What signals the cellular machinery uses to determine where to localize miRNA-target pairs for translational repression is unclear. There appear to be no consistent differences between the miRNA hairpin secondary structures and miRNA/miRNA* duplexes of AMP1-dependent and AMP1-independent miRNAs (Fig. S2). Although it is perhaps unlikely that any such signals would persist during miRNA processing. The strength of target complementarity is known to affect silencing efficacy (Li et al., 2014), and could also drive sub-cellular distribution, but there is also no trend in target mismatch number between the AMP1-dependent/independent classes of miRNA (Table S1). Given the overlapping expression domains of a number of these miRNAs (reviewed in Fouracre and Poethig, 2016), it is unlikely that the site of translational repression is developmentally regulated. At the cellular level, there is evidence to suggest that miRNA sequences include signals that control the specificity of inter-cellular mobility (Skopelitis et al., 2018). It will be fascinating to see if the same signaling mechanisms determine the destination of miRNAs within cells.
Materials and Methods
Plant material and growth conditions
Col was used as the genetic background for all stocks. The following genetic lines have been described previously: amp1-1 (Chaudhury et al., 1993); SPL9::sSPL9-GUS, SPL9::rSPL9-GUS (Xu et al., 2016); 35S::MIM156 (Fouracre and Poethig, 2019); clv1-4 (Clark et al., 1993); MYB33::sMYB33-GUS, MYB33::rMYB33-GUS (Millar and Gubler, 2005). clv3-10 (CS68823) was obtained from the Arabidopsis Biological Resource Center (Ohio State University). Seeds were sown on fertilized Farfard #2 soil (Farfard) and kept at 4°C for 3 days prior to transfer to a growth chamber, with the transfer day counted as day 0 for plant age (0 DAP). Plant were grown at 22°C under a mix of both white (USHIO F32T8/741) and red-enriched (Interlectric F32/T8/WS Gro-Lite) fluorescent bulbs in either long day (16 hrs. light/8 hrs. dark; 80 μmol m−2 s−1) or short day (10 hrs light/14 hrs dark; 120 μmol m−2 s−1) conditions.
GUS staining
Plants were fixed in 90% acetone on ice for 10 minutes and washed with GUS staining buffer (5mM potassium ferricyanide and 5mM ferrocyanide in 0.1M PO4 buffer) and stained for between 8 hrs and overnight (depending on transgene strength) at 37°C in 2mM X-Gluc GUS staining buffer. For the quantification of GUS staining intensity, ~1mm LP were harvested at 21 DAP, stained O/N and images of stained primordia converted from RGB color mode to hue saturation brightness mode as previously described (Béziat et al., 2017). A consistent position in the middle of the leaf lamina, adjacent to the midvein, was used for measurement.
Histology
Shoot apices were cleared and imaged according to a described protocol (Chou et al., 2016).
RNA expression analyses
Tissue (either shoot apices with leaf primordia ≤1mm attached or isolated leaf primordia 0.5-1mm in size – as specified in the text) were ground in liquid nitrogen and total RNA extracted using Trizol (Invitrogen) as per the manufacturer’s instructions. RNA was DNAse treated with RQ1 (Promgea) and 250ng-1μg of RNA was used for reverse transcription using Superscript III (Invitrogen). Gene specific RT primers were used to amplify miR156, miR157, miR172 and SnoR101 and a polyT primer for mRNA amplification. Three-step qPCR of cDNA was carried out using SYBR-Green Master Mix (Bimake). qPCR reactions were run in triplicate and an average taken. For analyses of amp1 shoot apices and clv mutants, separate RNA extractions of three biological replicates were carried out. For analyses of amp1 leaf primordia, three reverse-transcription replicates from single RNA extractions were carried out for each sample (at least 60 LP were pooled for each RNA extraction). 8 DAP samples were collected twice - once as part of a biological replicate with 13-13 DAP and once as part of a biological replicate with 20 DAP samples. Relative transcript levels were normalized to snoR101 (for miRNAs) and ACT2 (amp1 shoot apices, clv mutants) or UBQ10 (amp1 leaf primordia) (for mRNAs) and expressed as a ratio of expression to WT (amp1 shoot apices, clv mutants) and WT 8 DAP (amp1 leaf primordia) samples
For the quantification of transcript cleavage, a modified 5’RACE protocol was followed as previously described (He et al., 2018). The data presented are the average of three ratios from separate reverse transcription replicates (six in the case of amp1 8 DAP – three reverse transcription replicates from two biological replicates).
The qPCR primers used in this study are listed in Supplementary Table 2.
Statistical analyses
A two-tailed Student’s t-test was used to carry out pairwise comparisons between different genotypes. For comparison of multiple samples, to decrease the chance of false positives, a one-way ANOVA followed by a Tukey test was used for multi-way comparisons. Statistical analyses were carried out in R (r-project.org) and Excel (Microsoft).
For figures featuring boxplots, boxes display the IQR (boxes), median (lines), and values beyond 1.5* IQR (whiskers); mean values are marked by a solid diamond (♦).
Competing interests
No competing interests declared
Funding
This work was supported by the National Institutes of Health grant R01-GM51893 to R. S.P.
Figure Legends
Supplementary Fig. 1. amp1 cauline leaves produce abaxial trichomes. Scale bar: 2mm
Supplementary Fig. 2 Predicted hairpin structures for miRNAs that are AMP1-dependent and independent for translational repression. AMP1-independent (this study) – miR156, miR157, miR159; AMP1-dependent (Li et al., 2013) – miR164, miR165, miR166 and miR398. Representative functional members of miRNA families are displayed. Stem-loop sequences were downloaded from miRBase (www.mirbase.org) and hairpin structures predicted using the default settings on RNAfold (http://rna.tbi.univie.ac.at/). Minimum free-energy models of hairpins are shown, color coded for base pair probability. Black lines are drawn alongside mature miRNA sequences.
Acknowledgements
We thank Anthony Millar (Australian National University) for the kind gift of the MYB33- GUS reporters, members of the Poethig lab for useful discussions and Melissa Morrison for assistance with collecting phenotypic data.