Abstract
Plant architecture is a main determinate of crop yield, and lateral branching significantly influences the number of inflorescences and seeds. The mechanism of axillary bud initiation remains unclear. This work aimed to examine how miRNAs regulate axillary bud initiation. By constructing a small RNA library and screening a mutant population, we revealed the initiation of axillary buds is specifically induced by miR394 and repressed by its target, LEAF CURLING RESPONSIVENESS (LCR). Using promoter-driven fluorescent tags and in situ hybridization, we showed that miR394 is localized in the center of the leaf axil where AMs are initiated. Through molecular and genetic research, we revealed that miR394/LCR may regulate REVOLUTA (REV) and SHOOT MERISTEMLESS (STM) to establish the axillary meristem. Immunoprecipitation-mass spectrometry studies revealed that LCR, as an F-box protein, may interact with TOPLESS (TPL) proteins and participate in ubiquitinated protein degradation. Our results reveal an important mechanism by which the miR394-regulated LCR accelerates the degradation of TPL to precisely modulate axillary bud initiation.
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Introduction
The axillary meristem (AM) is a highly dynamic and continuously active stem cell system located in leaf axils and is responsible for the generation of lateral shoots in plants1,2,3. AM development includes initiation and bud outgrowth4. During AM initiation, a morphologically detectable bump emerges from the axils of leaves and develops into a bud5,6. Little is known about AM initiation due to its subtle structure. To date, several transcription factors (TFs) that regulate AM initiation have been characterized6,7,8,9,10,11,12,13,14.
As AMs initiate at the boundaries between the SAM and young leaf primordia, some transcription factors, including CUP SHAPED COTYLEDON (CUCs)9, LATERAL SUPPRESSOR (LAS)8 and REGULATOR OF AXILLARY MERISTEMS (RAXs)13, enriched in the boundaries specify boundary formation and affect AM initiation. AM initiation requires a meristematic cell population that continuously expresses the marker gene STM6,10,14. STM expression is dynamic and associated with two phases of stem cell division. In the first phase, in leaf axils younger than P9 (the ninth earliest leaf primordium), STM is expressed at a low level that is sufficient for stem cell competence but not for AM initiation10. In the second phase, STM is highly expressed in leaf axils older than P10, which promotes the formation of AMs10. REV, which belongs to the HD-ZIPIII family targeted by miR165/166, is expressed co-ordinately with AM initiation and is the only member of this family that can activate stem cells during AM initiation5,15,16. The level of REV is kept low on the adaxial side of young leaf axils (<P10) and increases in the old leaf axils (>P11) when AMs initiate14,17. REV was found to promote STM directly by binding the STM promoter to activate AM initiation10. After the induction of STM and gain of meristem activity, the homeodomain transcription factor WUSCHEL (WUS) promotes AM initiation around P13 leaf axils11. WUS expression is also feedback regulated by CLAVATA3 (CLV3) in AMs12. Auxin and cytokinins are two major hormones that regulate meristem development18. In boundaries and AMs, these two hormones, along with brassinosteroids (BRs), also play vital roles11,17,19,20. In some cases, TFs in these hormonal pathways combine with general transcription repressors, such as TPL, to precisely regulate meristem marker genes21,22,23,24,25.
MicroRNAs (miRNAs), endogenous small noncoding RNAs ranging in length from 20 to 24 nucleotides, play critical roles in plant development26,27,28,29,30,31. miRNAs control target gene expression at the posttranscriptional level by directly cleaving target mRNAs or inhibiting their translation32,33. Although the ability of miR165/166 to cleave REV and the role of the miRNA effector ARGONAUTE10 (AGO10) in AM initiation have been reported17,34,35, whether other miRNAs participate in AM development and how they function are unknown.
miR394 is a highly conserved miRNA of 20 nucleotides that was first identified in Arabidopsis thaliana36,37. Previous studies have reported that miR394 is involved in regulating meristem development, leaf morphogenesis, and stress resistance pathways38,39,40,41,42,43,44,45. In the shoot apical meristem, miR394 acts as a mobile signal produced from the protoderm and moves to the underlying cells to maintain stem cell activity by balancing the loop of WUS-CLV3. In its destination, the organizing centre (OC), miR394b inhibits the transcript levels of its target Leaf Curling Responsiveness (LCR)39. Furthermore, LCR may associate with the MLP-LIKE PROTEIN 28 (MLP28) family member to regulate SAM development41. However, the involvement of miR394 and LCR in AM development has not been reported, and the pathways affected by miRNAs and F-box proteins remain intriguing.
In this study, we constructed a small RNA library using boundary enrichment tissues, screened miRNAs potentially involved in AM development, and discovered that miR394 was highly expressed in AMs. We found that miR394/LCR expression exhibited temporal and spatial patterns that closely matched the dynamics of AM initiation: miR394 inhibited LCR in the adjacent domain between the leaf adaxial side and boundary, where AMs initiate. The inhibition continued throughout AM initiation within the buds. Moreover, we found that LCR down-regulated STM and REV expression. In addition, LCR acts as a specific Skp1/Cullin1/F-box (SCF) E3 ligase complex and targets TPL, which functions as a positive regulator of AM initiation. Collectively, our findings identified three modulators of AM initiation and established a pathway mediated by miR394 and F-box proteins in AMs.
Results
miR394 promoted and LCR inhibited AM initiation in Arabidopsis
To identify miRNAs involved in axillary meristem formation, we sampled seedlings from Col-0 plants grown under short-day conditions for 10 days (had only 2–4 leaves and almost no AMs had formed) and boundary enrichment tissues (vegetative shoot apex with leaf removal) from Col-0 plants grown under short-day conditions for 30 days (approximately 26 to 30 leaves emerged, at which time a large number of AMs were initiated in leaf axils), individually. Small RNA sequencing (sRNA-seq) were used to profile miRNA expression and we compared the sRNA-seq data of the vegetative shoot apex with that of the seedlings. Ultimately, we identified 46 up-regulated and 39 down-regulated high-confidence differentially expressed (DE) miRNAs (FDR < 0.5) (Supplementary Data 1). It was worth noting that among these DE miRNAs, miR394a and miR394b were highly enriched with great significance at the boundaries (Fig. 1a). Previous studies have shown that mir394b-1 produces “pin-like” shoots in the ago10-1 background, which is indicative of stem cell defects in Arabidopsis39,46. By screening a small RNA-related mutant population, we confirmed that AMs consistently failed to form in many rosette leaf axils of mir394b-1 but normally formed in every leaf axil of the wild type (Fig. 1b). To understand how miR394 regulates the formation of AMs in rosette leaf axils in more detail, we knocked out miR394a and miR394b via CRISPR/Cas9 in mir394a-cr and mir394b-cr lines and overexpressed miR394a and miR394b in miR394A-OX and miR394B-OX lines (Supplementary Fig. 1a–c). After genomic identification, three independent homozygous lines for each genotype were selected for further study (Supplementary Fig. 1d–f). We found that both the mir394a-cr lines (#1 delete 1 bp, #2 delete 44 bp and #3 delete 45 bp) and the mir394b-cr lines (#1 delete 2 bp, #2 delete 28 bp and #3 add 1 bp) presented AM defects similar to but less severe than those of mir394b-1 (Fig. 1b and Supplementary Fig. 2a). We further quantified AMs of the mir394a/b double mutant and observed many fewer AMs than in the single mutants (Fig. 1b, c), suggesting that miR394a and miR394b may act redundantly in controlling AM formation. In contrast, compared with the WT, miR394-OX did not result in a change in the number of AMs (Supplementary Fig. 2a).
a Volcano plots showing differentially expressed (DE) miRNAs in SAMs and AMs compared with whole seedlings determined by sRNA-seq. The red dots represent miRNAs whose expression was significantly up-regulated, the blue dots represent miRNAs whose expression was significantly down-regulated, and the gray dots represent miRNAs whose expression was not significantly changed. b Schematic representation of axillary bud formation. Compared with the WT, the mir394a-cr, mir394b-1, mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m transgenic lines presented different degrees of AM defects in early leaves. The thick black horizontal line represents the border between the youngest rosette leaf and the oldest cauline leaf. Each column represents a single plant, and each square represents an individual leaf axil. The bottom row represents the oldest rosette leaf axils, with progressively younger leaves above. Green indicates the presence of an axillary bud; yellow indicates the absence of an axillary bud. All the plants were grown under short-day conditions for 30 days and then shifted to long-day conditions for 30 days to count the number of axillary buds. c Morphology of rosette leaf axils in WT, mir394a/b mutant, pLCR::LCR4m or p35S::LCR4m transgenic lines. AM was visible in the WT (white dotted circle) but absent in the other genotypes (yellow dotted circle) under a dissecting microscope. All the plants were grown under short-day conditions for 30 days and then subjected to long-day conditions for 30 days. Scale bars, 1 mm. d Scanning electron micrographs of the basal regions of P15 rosette leaves showing AM formation (white dotted circle) in WT but the absence of AMs (yellow dotted circle) in the mir394a/b mutant or LCR4m overexpression lines. The plants were grown under short-day conditions for 21 days. Scale bars, 20 μm. e Cross sections of vegetative shoot apex were stained with toluidine blue O. Images show a dome-shaped AM in the leaf axil of P15 in WT (arrows) but not in the mir394a/b mutant or LCR overexpression lines (yellow arrows). The plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. At least three samples were analyzed with similar results for each experiment in (c–e).
To determine whether LCR gene, which is a known miR394 targets, is the cause of AM defects36, quantitative real-time PCR (qRT-PCR) was used to measure LCR mRNA in the miR394 mutants. LCR was expressed at much higher levels when miR394a and miR394b were knocked down in the mir394a/b double mutant (Supplementary Fig. 1d, e). To analyze whether LCR inhibits AM formation, we obtained a loss-of-function mutant of LCR, lcr-1 (SALK_136833), with a T-DNA insertion and generated lcr-cr lines via CRISPR/Cas9 (Supplementary Fig. 3a, b). After PCR confirmation, we obtained three independent homozygous lcr-cr mutants (#1 delete 6 bp, #2 delete 21 bp and #3 add 1 bp) for further study. Both the lcr-1 and lcr-cr mutants presented reduced LCR mRNA levels (Supplementary Fig. 3c, d) and normal AM formation (Supplementary Fig. 2a). Moreover, we expressed LCR and the miR394-resistant versions LCR4m39 and LCR5m43 in Col-0, driven by the LCR promoter or the CaMV 35S promoter (Supplementary Fig. 4a). Genotyping combined with qRT-PCR and immunoblot analyses confirmed that we had obtained more than three independent LCR-OX and LCR4m-OX homozygous lines (Supplementary Fig. 4b–e). As expected for the miR394 targets, LCR mRNAs were highly accumulated in pLCR::LCR4m and p35S::LCR4m (Supplementary Fig. 4c), leading to a strong AM deficiency phenotype (Fig. 1b, c). We carefully analyzed AM development in p35S::LCR4m, whose phenotype was more severe, and found that 97.7% of rosette leaves presented bare axils. Among cauline leaf axils, 71.4% were without AMs, 14.3% had terminated buds, and 14.3% had filamentous structures. In addition, p35S::LCR4m also presented defects in floral primordia (Supplementary Fig. 5a–o). In contrast to the LCR4m-OX plants, LCR loss-of-function mutants grew faster and formed normal AMs in rosette leaves (Supplementary Fig. 2a). Notably, the AM defect of mir394a/b was rescued by crossing with lcr-1 (Supplementary Fig. 2a), confirming that miR394 promotes AM formation through the downregulation of LCR mRNA.
We observed the AM morphology of the mir394a/b double mutant, pLCR::LCR4m, p35S::LCR4m and WT via scanning electron microscopy (SEM). While the WT had obvious dome-shaped bulges at the adaxial base of P15, the mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m lacked proliferating cells at the leaf axils at the same stage (Fig. 1d). We observed anatomical changes in the leaf axils through tissue sectioning. In the WT, more densely stained cells formed morphologically distinguishable bumps at stages P14 to P16. In the mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m, the frequencies of densely stained cells and AM structures were highly reduced in the leaf axils at the same stages (Fig. 1e). Taken together, these data indicate that miR394 expression in the leaf axil is necessary for AM initiation.
LCR encodes a conserved F-box protein47,48,49 (Supplementary Fig. 6a–d and Supplementary Data 2). To confirm whether LCR affects AM initiation through the F-box domain, we constructed overexpression lines in which the F-box domain was removed (Supplementary Fig. 4a–e). We tracked AM development in LCRΔF-box-OX and LCR4mΔF-box-OX plants and found that most leaf axils formed normal AMs (Supplementary Fig. 2a–e). These results indicate that LCR may act as a negative regulator of AM initiation through its F-box domain.
miR394 and LCR are expressed on the adaxial side of leaves and AMs
AMs are initiated at the boundary zone in the shoot apex during vegetative development. AM initiation is regulated temporally and spatially, and a morphologically distinguishable structure is formed by P11. We therefore tested whether miR394 is expressed in the shoot apex. To visualize the real-time dynamics of miR394 expression, we constructed pMIR394A::GFPer and pMIR394B::GFPer reporter lines (Supplementary Fig. 7a, b) and tracked miR394 transcription in young leaf axils. We found that MIR394A had a strong signal in the boundary region between the emerging leaf primordia and the SAM, and this signal spread into the adaxial domain of the leaves as well but was lower in AMs (Fig. 2a and Supplementary Fig. 8a). In addition, a similar pattern of MIR394B expression was observed in primordia and boundary cells as that of MIR394A, but the signals in AMs were relatively greater than those in MIR394A (Fig. 2b and Supplementary Fig. 8b). RNA in situ hybridization assays revealed that mature miR394 accumulated in the SAM, AMs, vasculature, and leaf primordia (especially the adaxial domain) (Fig. 2c). Through tracking of miR394 at boundaries and AMs, we found that miR394 was enriched in the upper cell layers and the center of the leaf axils where AMs initiate (phase 1–2) and was continuously expressed throughout the initiation of AMs (phase 3–9) (Supplementary Fig. 9a–p). These results indicated that miR394a and miR394b were co-expressed at the boundary but partially overlapped in AMs, implying the overlapping and distinguishing roles of the family members.
a, b Longitudinal and transverse sections through the vegetative shoot apex of pMIR394A::GFPer and pMIR394B::GFPer. MIR394A (a) and MIR394B (b) signals are shown in green, and the autofluorescence of chlorophyll is shown in red. c In situ hybridization revealed the expression patterns of mature miR394. d Expression of LCR (green) in the shoot apex. Sections were mix with autofluorescence (red). e LCR mRNA accumulation in the shoot apex was determined via RNA in situ hybridization in pLCR::LCR4m. Confocal images were taken of agarose sections. The asterisks represent the SAM, the arrows represent AMs, and the dashed lines represent the leaf axils where AMs were initiated. The numbers in the upper right corner represent the relative distances from the shoot apex. All the plants were grown under short-day conditions for 30 days. AS, antisense probes; S, sense probes. Scale bars, 100 μm. At least three samples were analyzed with similar results for each experiment in (a–e).
To investigate the expression patterns of LCR during AM initiation, we constructed LCR and LCR4m reporters by fusing the CDS to GFP under the native LCR promoter, but no stable LCR signal was captured, even with F-box removal. Therefore, referring to a previous report39, we amplified a fragment containing the LCR promoter, the first exon, and three amino acids from the second exon and fused it to GFP to form an miR394-resistant transcriptional reporter of gLCR::GFP (Supplementary Fig. 7a, b). Using this reporter line, we found that GFP was strongly enriched in the adaxial domain of leaf primordia, boundaries, SAM and AMs (Fig. 2d and Supplementary Fig. 8c). In addition, although endogenous and exogenous LCR was not detected in pLCR::LCR via in situ hybridization, the miR394-resistant version of pLCR::LCR4m presented a similar mRNA distribution in leaf axils to that of the gLCR::GFP reporter (Fig. 2e), suggesting that miR394 inhibits LCR post transcriptionally in leaf axils.
To further confirm the role of the LCR protein in leaf axils, we specifically expressed LCR5m, driven by the abaxial domain-specific promoter KANADI (KAN1) or the adaxial domain-specific promoter REV (Supplementary Fig. 7a, b). We found that pREV::LCR4m presented a phenotype similar to that of pLCR::LCR4m, with severe defects in AMs, but that of pKAN1::LCR4m remained the same as that of the WT (Supplementary Fig. 10a–c). It is known that miR394 inhibits LCR transcript levels, but it remains to be determined whether miR394 inhibition of LCR is required for AMs initiation (Fig. 2 and Supplementary Fig. 11a–d). Transgenic plants harboring p35S::LCR-GFP and p35S::LCR4m-GFP were obtained, and nuclear signals were verified in the root tips (Supplementary Fig. 11e, i). While strong LCR4m signals were observed in the shoot apex, only a weak LCR signal was captured in cells below the SAM (Supplementary Fig. 11f–h, j–l). Moreover, western blot analysis confirmed that the LCR4m protein was significantly more highly expressed than the LCR protein (Supplementary Fig. 11q, r), confirming that the expression of LCR was strictly controlled by miR394 in the shoot apex. In addition, after removing the F-box domain, LCRΔF-box still presented similar signals in root nuclei as the full-length protein (Supplementary Fig. 11m), and a weak signal appeared in the adaxial domain of the leaves and AMs (Supplementary Fig. 11n–p), indicating that the level of LCRΔF-box protein also increased (Supplementary Fig. 11q, r). By treating LCR4m with MG132 (a 26S proteasome inhibitor), we also found that the degradation rate of LCR4m decreased (Supplementary Fig. 11s). These results suggest that LCR may be degraded through the 26S proteasome.
miR394 activated the REV and STM pathways during AM initiation
To explore the pathways affected by miR394/LCR during AM initiation, we analyzed the transcriptomes of miR394A-OX, mir394a-cr, mir394b-1, pLCR::LCR4m and WT (Supplementary Data 3). We conducted RNA sequencing of vegetative shoot apex of plants grown under short-day conditions for 30 days. After strict screening with a cutoff of Log2 (fold change >1) and p adjusted (FDR) < 0.05, we identified 2224 differentially expressed genes (DEGs) in the miR394A-OX lines, 3002 DEGs in the miR394a-cr mutants, 2322 DEGs in the miR394b-1 mutants and 2539 DEGs in the pLCR::LCR4m lines (Supplementary Fig. 12a). Gene Ontology (GO) terms related to post-embryo and leaf development were enriched in the miR394 mutant and LCR4m-OX lines, respectively (Supplementary Fig. 12b). We next used the k-means clustering algorithm to cluster all expressed genes, which were categorized into nine co-expression modules. Notably, LCR4m-OX displayed a more similar profile to the miR394b-1 mutant than did miR394a-cr, characterized by pathways including photosynthesis (Cluster 2) and leaf development (Cluster 8) (Supplementary Fig. 12c).
Previous reports have shown that AM initiation requires cells to continuously express STM and that REV can directly activate STM expression10. Subsequently, cytokinin activates WUS expression de novo to establish AMs11. Thus, we examined REV, STM, WUS and CLV3 expression in miR394/LCR mutants via qRT-PCR. The results revealed that REV, STM and CLV3 expression levels were lower in the mir394a/b mutants and LCR4m-OX plants than in the WT plants (Supplementary Fig. 12d). However, the difference in WUS expression was not significant (Supplementary Fig. 12d). To investigate the expression patterns of REV and STM in vivo, we crossed the marker lines pSTM::STM-Venus50 and pREV::REV-Venus20 with the p35S::LCR4m transgenic lines. In young leaf axils (<P9), the STM signals persisted at the boundaries and SAM in the WT (Fig. 3a). The STM signals also remained at the p35S::LCR4m boundaries and SAM but were much weaker than those of the WT (Fig. 3b). Starting at P9, the STM signals in the older leaf axils of p35S::LCR4m weakened and even completely disappeared at P13 (Fig. 3b), whereas STM was evidently up-regulated in the WT (Fig. 3a). The low expression of STM may explain the deficiency of AMs in LCR4m-OX plants. We next asked whether REV expression is affected in p35S::LCR4m plants. In the WT, REV-Venus signals were present in AMs (Fig. 3c). However, REV was hardly detectable in AMs or on the adaxial side of the leaf primordia in the p35S::LCR4m plants (Fig. 3d). To monitor AM initiation quantitatively, we performed live imaging of leaf axils isolated from the shoot apex and grown under short-day conditions for 17 days. We observed small amounts of axillary bud formation in detached axils of LCR4m-OX from P8/9 and P10/11. Then, we performed time-course live imaging with detached P10/11 axils to closely trace STM and REV expression and AM initiation (Supplementary Fig. 13a, c, e, g). After 24 h of cultivation in growth medium, most axils of LCR4m-OX plants lost STM and REV expression, and cell proliferation stopped, whereas the number of STM-expressing cells in the axils of WT plants dramatically increased and AMs emerged (Supplementary Fig. 13b, d, f, h). To determine whether the loss of STM and REV expression in the leaf axil was responsible for the AM initiation defects, we individually introduced inducible STM-GR (glucocorticoid receptor) under the 35S promoter and REV-GR under the REV promoter into LCR4m-OX. We performed dexamethasone (DEX) induction on pREV::REV-GR-HA p35S::LCR4m and p35S::STM-GR p35S::LCR4m plants and found that the formation of AMs was restored in both plants (Supplementary Fig. 13i–l). These observations confirmed that the inhibition of LCR by miR394 is necessary for promoting STM and REV expression during AM initiation.
Continuous transverse sections and longitudinal sections showing pSTM::STM-Venus (green) expression and chlorophyll autofluorescence (red) in the shoot apex of the WT (a) and p35S::LCR4m (b) plants. STM was activated in AMs of WT plants (white arrows) but maintained low expression levels in the leaf axils of p35S::LCR4m plants (yellow arrows). Continuous transverse sections and longitudinal sections of pREV::REV-Venus (c) and pREV::REV-Venus p35S::LCR4m (d) shoot apex. REV (green) signals were strong in the AMs (white arrow) of the WT but were extremely weak in the leaf axils of p35S::LCR4m (yellow arrows). The asterisks represent the SAM, the arrows represent AMs, and the dashed lines represent the leaf axils where AMs were initiated. The numbers in the upper right corner represent the relative distances from the shoot apex. All the plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. At least three samples were analyzed in (a–d) with similar results for each experiment.
LCR is associated with TPL and mediates ubiquitin modification
To gain further insight into the role of LCR in AM initiation, we evaluated the LCR interactome via immunoprecipitation-mass spectrometry (IP-MS) and identified potential substrates (Supplementary Data 4). We used GFP-trap and protein extracts from p35S::LCR-GFP, p35S::LCR4m-GFP and p35S::GFP transgenic plants for immunoprecipitation. In total, seven significant proteins were selected in two biological replicates (Fig. 4a). These included TOPLESS (TPL), SERRATE (SE), HNRNP R-LIKE PROTEIN (HRLP), DOT2, NITRILASE 2 (NIT2), SUPPRESSOR OF MAX2 1 (SMAX1) and AUXIN RESISTANT 6 (AXR6). Among them, TPL was previously characterized as a transcriptional corepressor that interacts with WUS to regulate SAM maintenance21,51. Notably, TPL and LCR were colocalized in the nuclei of the leaf protoplasts (Supplementary Fig. 14d). We also obtained p35S::TPL-mCherry p35S::LCRΔF-box-GFP transgenic plants and observed colocalized signals in the nuclei of the roots (Supplementary Fig. 14a–c, e). We thus tested whether LCR can interact with TPL. As expected, yeast two-hybrid (Y2H) assays revealed that LCR proteins strongly interact with TPL (Fig. 4b). Domain deletion analysis revealed that the C-terminus of LCR (164-467 aa) was required for the interaction (Fig. 4b). Moreover, truncated TPL containing only aa 93 to 352 could interact with LCR (Supplementary Fig. 15a, b). Furthermore, bimolecular fluorescence complementation (BiFC) using LCR/LCRΔF-box-cYFP and TPL-nYFP in Nicotiana benthamiana epidermal cells confirmed that LCR physically interacted with TPL in nuclei in vivo (Fig. 4c). To confirm the LCR-TPL interaction in Arabidopsis, we generated p35S::LCR4m-GFP transgenic plants and performed a coimmunoprecipitation (Co-IP) assays. Consistent with the above results, LCR coimmunoprecipitated with endogenous TPL (Supplementary Fig. 14f). Arabidopsis expresses five TOPLESS RELATED (TPR) proteins in total, namely, TPL, TPR1 (TOPLESS-RELATED 1), TPR2 (TOPLESS-RELATED 2), TPR3 (TOPLESS-RELATED 3) and TPR4 (TOPLESS-RELATED 4)21. Therefore, we tested the potential interaction between LCR and TPL homologs via Y2H and found that LCR may interact with TPR1, TPR2 and TPR4 (Supplementary Fig. 15c, d).
a IP-MS using p35S::LCR-GFP and p35S::LCR4m-GFP as baits to identify LCR-interacting proteins in Arabidopsis thaliana. Important proteins are shown in the list. p35S::GFP served as the negative control. b The interaction between LCR and TPL was verified via Y2H assays. The protein domains of LCR and TPL used in Y2H are shown at the top. Yeast cells transformed with BD-LCR, BD-LCRF-box and BD-LCRΔN/AD-TPL were grown and turned blue on SD-L/-T/-H/-A + X-α-gal medium. Yeast transformed with BD-LCRΔC/AD-TPL or BD-LCR/AD-Blank did not grow on SD-L/-T/-H/-A + X-α-gal medium. pGADT7-T and pGBKT7-53 were used as positive controls, whereas empty vectors were co-transformed as negative controls. c BiFC assays revealed that LCR and LCRΔF-box interacted with TPL in vivo in N. benthamiana (tobacco) leaves. H2B-mCherry is a nuclear-localized marker. The combination of 35S::cYFP and 35S::TPL-nYFP was used as a negative control. Scale bars, 5 μm. d, e Stability test of TPL in the WT and lcr-1 backgrounds. Total protein was extracted from p35S::TPL-GFP (blue) and p35S::TPL-GFP lcr-1 (red) plants with or without MG132 treatment. TPL degradation was measured by immunoblotting with an anti-TPL antibody at the indicated time points. Actin was used as a loading control. Band intensities were quantified with ImageJ in (e). Data are mean ± s.d. (***p < 0.001, ns no significant difference, two-way ANOVA with Tukey’s honestly significant difference test. n = 3 biological replicates). The experiments in (b–e) were performed at least three times with similar results.
We next investigated whether the expression and/or protein levels of TPL were altered by LCR. Compared with those in the WT, TPL and TPR1 were slightly down-regulated and TPR3 was slightly up-regulated in pLCR::LCR4m, but the expression of TPR2 and TPR4 was not significantly affected by pLCR::LCR4m (Supplementary Fig. 16a). To analyze TPL protein levels, we individually transformed p35S::TPL-mCherry into WT and p35S::LCR4m-GFP plants (Supplementary Fig. 14a–c). Immunoblot analysis revealed obviously reduced TPL-mCherry levels in the LCR4m background (Supplementary Fig. 16b, c). Moreover, LCR4m-GFP abundance was also significantly lower in p35S::LCR4m-GFP p35S::TPL-mCherry transgenic plants than in p35S::LCR4m-GFP transgenic plants (Supplementary Fig. 16b, c). To reinforce this conclusion, we also observed the fluorescence signals of p35S::TPL-mCherry, p35S::LCR4m-GFP and p35S::LCR4m-GFP p35S::TPL-mCherry in roots and shoots. Both the LCR and TPL signals were drastically reduced in the roots but absent from the shoots of p35S::LCR4m-GFP p35S::TPL-mCherry (Supplementary Fig. 16d).
LCR plays a major role in the Skp1/Cullin1/F-box (SCF) E3 ligase complex47 and performs its ubiquitination function through the F-box domain (Supplementary Fig. 6). Thus, we hypothesized that LCR may be able to ubiquitinate TPL. To test this hypothesis, we overexpressed TPL-GFP in WT and lcr-1 (Supplementary Fig. 14a–c) and performed immunoprecipitation with an anti-GFP antibody. Subsequent immunoblot analysis of the IP proteins via anti-Ubi antibodies revealed that TPL was ubiquitinated in planta. The level of TPL ubiquitination was lower in lcr-1 than in WT (Supplementary Fig. 16e). Previous studies revealed that most ubiquitylation of target proteins is associated with 26S proteasome-mediated degradation25,48. To confirm whether TPL degradation was mediated by the 26S proteasome, we prepared total protein extracts from p35S::TPL-GFP and p35S::TPL-GFP lcr-1 seedlings at the indicated time points. We found that TPL in protein extracts from lcr-1 plants degraded more slowly than TPL protein from WT plant extracts did. The addition of the proteasome inhibitor MG132 further decreased the rate of TPL degradation (Fig. 4d, e). These results indicated that the LCR and TPL proteins physically interact with each other and that LCR may negatively regulate TPL abundance via protein degradation.
TPL is expressed in leaf axils and promotes AM initiation
We were curious to know whether TPL regulates AM initiation. For this purpose, we carefully observed the AM phenotype of the tpl mutants. The strong allele tpl-1 with a point mutation (N176H)21 acts as a dominant negative mutant for multiple TPR family members and displays severe shoot defects and the absence of normal AMs (see below and Supplementary Fig. 18a). Therefore, we obtained another moderate mutant, tpl (SALK_097230), with a T-DNA inserted in the WD40 domain of TPL. However, unfortunately, tpl did not result in any change in AMs (Supplementary Fig. 17a, b). Considering that TPL/TPR family members share conserved structures and redundantly regulate apical fate during embryogenesis21,23,25, we inferred that TPL/TPR family proteins may also function redundantly in AM initiation. Therefore, we observed AMs in the tpr1, tpr2, tpr3, tpr4 mutants and the tpl tpr1 tpr4 triple mutant. Notably, AMs were not affected in the single mutants but were severely compromised in the triple mutant (Fig. 5a and Supplementary Fig. 17a, b). In support of these observations, we carefully analyzed this defect and found that it originated in tpl tpr1 tpr4 triple mutant leaf axils (Fig. 5b, c). Anatomical analysis revealed that densely stained cells formed morphologically distinguishable bumps in the WT but were lacking in tpl tpr1 tpr4 (Fig. 5d–g). Moreover, plants that overexpressed TPL normally formed one AM per leaf axil (Fig. 6a, b and Supplementary Fig. 17a, b). To monitor TPL expression during AM initiation, we constructed pTPL::GFP marker lines and observed strong fluorescence signals in the boundary region, AMs and SAM (Fig. 5h and Supplementary Fig. 14a, b). We also analyzed the expression patterns of other TRR proteins via in situ hybridization. As expected, TPL mRNA was widely expressed in the shoot apex and young leaf primordia (more extensively than the position of AM initiation) and was particularly strongly expressed in the SAM, AMs and vasculature, which was consistent with pTPL::GFP (Fig. 5i and Supplementary Fig. 17c, g). TPR1 and TPR4 were detected in the leaf primordia, vasculature, SAM and AMs and largely overlapped with TPL (Fig. 5i and Supplementary Fig. 17d, h, f, j). In contrast, TPR2 was specifically present in the vasculature, boundaries and centers of the SAM and AMs (Fig. 5i and Supplementary Fig. 17e, i). The expression patterns of TPL/TPR family proteins are consistent with their overlapping roles in controlling AM initiation. Moreover, we examined the accumulation of STM and REV in tpl tpr1 tpr4 via in situ hybridization. REV and STM were clearly present in the AMs of WT plants but nearly undetectable in the tpl tpr1 tpr4 triple mutant (Fig. 5j and Supplementary Fig. 17k–n).
a Schematic representation of AMs in the WT and the tpl tpr1 tpr4 triple mutant. The diagram is as described in Fig. 1b. Morphology of AMs in the WT (b) and the tpl tpr1 tpr4 triple mutant (c). The plants were grown under short-day conditions for 30 days and then transferred to long-day conditions for 30 days. Scale bars, 1 mm. d–g Cross- and longitudinal sections of shoot apex of WT and the tpl tpr1 tpr4 triple mutant stained with toluidine blue O. A dome-shaped AM was visible in the WT (arrows, d–f) but not in the tpl tpr1 tpr4 triple mutant (yellow arrows, e–g). All the plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. h Expression patterns of pTPL::GFP (green) in the vegetative shoot apex. TPL is expressed in leaf axils (dotted white lines), AMs (arrows) and SAMs (asterisks). The numbers in the upper right corner represent the relative distances from the shoot apex. The plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. i RNA in situ hybridization was used to determine the expression patterns of TPL, TPR1, TPR2 and TPR4 in the vegetative shoot apex of Col-0. Asterisks indicate SAM, arrows indicate AMs, and dotted lines indicate leaf axils. All the plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. j Transverse sections through a vegetative shoot apex showing the distribution of STM and REV mRNAs in the WT and the tpl tpr1 tpr4 triple mutant. STM accumulated in the SAM and AMs (black arrows) of the WT but not in the AMs of the tpl tpr1 tpr4 triple mutant (yellow arrows). REV was found in the AMs of the WT (black arrows), but no signal was detected in the AMs of the tpl tpr1 tpr4 triple mutant (yellow arrows). All the plants were grown under short-day conditions for 30 days. Scale bars, 100 μm. At least three samples were analyzed in (b–j) with similar results for each experiment.
a Phenotypes of the WT, p35S::TPL, p35S::LCR4m, p35S::TPL p35S::LCR4m, tpl-1 and p35S::LCR4m tpl-1 plants grown for 30 days under short-day conditions. Scale bars, 3 cm. b Schematic representation of AMs in the WT, p35S::TPL, p35S::LCR4m and p35S::TPL p35S::LCR4m hybrid generations. The diagram is as described in Fig. 1b. c Venn diagram showing the number of overlapping DEGs between the LCR4m-OX and tpl tpr1 tpr4 mutants compared with the WT. The numbers of up-regulated (orange) and down-regulated (gray) DEGs are shown. The vegetative shoot of two biological replicates (n = 2) were collected at 30 days under short-day conditions for RNA sequencing. d Pearson correlation of common DEGs in LCR4m-OX and tpl tpr1 tpr4. The numerical values represent the log2 (FPKM+1) in the two samples. p values were determined via two-tailed Pearson correlation coefficient analysis. e A working model elucidating the crosstalk among miR394, LCR and TPL in AMs. miR394 is expressed in shoot apex and strictly inhibits LCR accumulation in leaf axils, to attenuate LCR mediated TPL degradation and activate REV and STM pathway, which is essential for AM initiation. Ab abaxial axis, Ad adaxial axis. The experiments in (a–d) were performed at least two times with similar results.
We next analyzed the genetic relationships between LCR and TPL in AM initiation. For this purpose, we crossed p35S::LCR4m with the tpl-1 mutant and obtained p35S::LCR4m tpl-1 plants (Supplementary Fig. 14c). The subsequent phenotyping revealed that the morphology of the p35S::LCR4m tpl-1 plants was similar to that of the tpl-1 plants, and the embryos failed to form apical meristems and showed varying degrees of cotyledon fusion (Fig. 6a and Supplementary Fig. 18a). In addition, we discovered that the overexpression of TPL rescued the AM deficiency of p35S::LCR4m, as reflected by the normal expression of REV and STM (Fig. 6a, b and Supplementary Fig. 18b). In support of these observations, we compared the transcriptomes of p35S::LCR4m and tpl tpr1 tpr4 in the vegetative shoot apex (Supplementary Data 5). Compared with those in the WT, we identified 1736 DEGs in p35S::LCR4m and 1324 DEGs in the tpl tpr1 tpr4 mutants (FDR < 0.05 and Log2 (fold change) >1). Ultimately, we identified 504 common genes with high confidence, comprising 239 (47.42%) up-regulated genes and 233 (46.23%) down-regulated genes (Fig. 6c). To better understand the relationships among the DEGs associated with p35S::LCR4m and tpl tpr1 tpr4, we analyzed the 504 DEGs via Pearson correlation analysis. Consistently, these common DEGs were highly correlated (R = 0.89) with respect to expression (Fig. 6d). These results indicated that LCR and TPL have similar effects on a common set of genes. Collectively, these results demonstrated that TPL may act downstream of LCR and as a target of LCR to positively regulate AM initiation via the REV-STM pathway (Fig. 6e).
Discussion
miRNAs play a vital role in plant development via the precise control of cell proliferation and differentiation26,27,31. However, almost no miRNAs have been characterized in AMs until now because of their subtle structure. AMs contain reactivated stem cells and various cell types, which make them representative of tissue development1,2,3. We resorted to a tissue-specific miRNA library and identified key regulators of AM initiation (Fig. 1). The number of cells expressing miR394 is limited in plants because miR394 expression has rarely been detected in libraries of small RNAs generated from whole seedlings. High levels of miR394 are expressed in boundary enrichment tissues, indicating a role of miR394 in the axillary meristem in addition to SAM. There are two copies of miR394 in Arabidopsis, of which miR394b plays a dominant role in SAM. Specifically, miR394b is transcribed in the L1 layer of the SAM and moves to the lower layers to execute a non-cell-autonomous role in stem cell maintenance by affecting WUS-CLV339. In AMs, miR394a and miR394b collaborate and play overlapping roles in promoting AM initiation, although the miR394b mutant displayed a more severe phenotype of AM deficiency (Fig. 1). MIR394A was strongly expressed in L3 and the underlying cells, whereas MIR394B was more highly expressed in L1-L3 of the AMs (Fig. 2). In situ hybridization revealed that mature miR394 was distributed in a gradient in AMs, with high expression levels in L1 and lower expression levels underneath (Fig. 2). In contrast, LCR showed the opposite expression pattern from L1 to L3 and in the cells underneath (Fig. 2). STM and REV expression in AMs indicated that miR394 levels persisted in leaf boundaries to maintain stem cells in younger leaf axils and increased to activate AM initiation in older leaf axils (Fig. 3). WUS did not show significant change in shoot apex of mir394 mutants may be due to tissue dilution. These results demonstrate that miR394 plays a conserved role in stem cell maintenance in both AMs and SAMs and promote meristem initiation in axils.
miRNAs cleave target mRNAs at the posttranscriptional level30,32,33. LCR is a reported target of miR394 and encodes an F-box protein36,44. Previous studies have shown the role of LCR in SAM and leaf development39,42,43,45. Here, genetic and cellular experiments revealed that LCR is expressed in AMs and negatively regulates AM initiation (Figs. 1–3). Furthermore, transgenic lines with truncated LCR indicated that the F-box domain is necessary for LCR function in AMs (Figs. 1 and 2). F-box proteins are well known to be tightly linked to protein ubiquitylation and degradation52; however, the substrates of the LCR protein are less well known. Here, we used IP-MS to screen LCR downstream substrates and identified TPL (Fig. 4). TPL was reported to control gene transcription with transcription factors as general repressors23,24,53. However, the role of TPL in AMs has not been studied. The phenotyping of tpl and homologous mutants revealed significant AM deficiency, similar to the LCR OX lines, indicating that this gene participated in AM initiation and coregulated common genes with LCR (Figs. 5 and 6). The expression of TPL in leaf axils and AMs verified that TPL directly regulates AM initiation in leaf axils (Fig. 5). Genetic data confirmed that LCR and TPL function downstream of miR394 (Fig. 6). Together, these results collectively reveal a pathway comprising miRNAs and F-box-mediated proteins that regulate AM formation.
Previous studies have focused on the transcriptional control of AMs, but the regulation of AMs at the protein level is less well understood7,8,10,12,13,14. AM initiation is a complicated process through which plants build architecture to reach the highest yield and respond to environmental conditions19. Here, we studied AM regulation at the protein level. LCR itself was strictly regulated in the shoot apex, as LCR signals were not easily detected in LCR OX and marker lines constructed with the native proteins. Only in mutated versions in which the miR394 binding sites were deleted or the F-box domain was deleted could LCR be detected in the transgenic lines. Nevertheless, the relationship between miR394 cleavage and F-box regulation, i.e., posttranscriptional and translational control of LCR, is not known. LCR, as an F-box protein, interacts with several important substrates related to certain pathways. TPL is a general transcription factor, and SE is a component of dicing bodies, which process small RNAs22,30. The interaction of LCR with other proteins, such as SE, deserves further study. In this study, we demonstrated that LCR interacted with TPL and accelerated TPL degradation, likely through ubiquitylation. The modification sites of the TPL and LCR proteins are also not clear. In addition to TPL, its functionally redundant homologs, TPR1, TPR2 and TPR4, also interact with LCR. Thus, LCR may also mediate the ubiquitylation and degradation of other TPR proteins; however, this possibility remains to be further validated. It has been reported that the regulation of PLETHORA1/2 (PLT1/PLT2) expression by TPL and HD-ZIPIII is necessary for apical/shoot fate in early embryogenesis54. In our study, we revealed that TPL and REV are also necessary for AM initiation confirming the positive roles of TPL and HD-ZIPIII in meristem development. Interestingly, the expression of REV was not observed in the leaf axils of the tpl tpr1 tpr4 triple mutant indicating that REV function downstream TPL and LCR in AM initiation. In brief, we provide a mechanistic framework to explain how plants regulate AM initiation at the transcriptional, posttranscriptional and protein levels. This framework not only includes a number of newly identified AM-defective mutants but also integrates the known genes STM and REV in the AM initiation pathway.
Methods
Growth conditions
Arabidopsis thaliana plants were grown at 22 °C under full-spectrum white fluorescent light under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions. For molecular identification and sRNA-seq in the seedlings, seeds were geminated on half-strength Murashige and Skoog (1/2 MS) medium and grown under short-day conditions for 10 days (2–4 leaves). For live imaging of the leaf axil regions, plants were grown under short-day conditions for 17 days (about 10–12 leaves). For scanning electron microscopy analysis of P15 axillary buds, plants were grown under short-day conditions for 21 days (14–16 leaves). For RNA-seq, sRNA-seq and quantitative analysis of mRNAs in the shoot apex, plants were grown under short-day conditions for 30 days (26–30 leaves). For sectioning and gene expression pattern analysis in the shoot apex, plants were grown under short-day conditions for 30 days before paraffin or low-melting point agarose sectioning. For AM phenotyping, plants were grown under short-day conditions for 30 days and then under long-day conditions for an additional 30 days to induce flowering before the axillary buds were counted.
Genetic material
Arabidopsis thaliana ecotypes Columbia (Col-0) and Landsberg erecta−0 (Ler) were used as wild-type controls. The mir394b-1 (single nucleotide mutation, G110A)39, tpl (SALK_097230)55, tpr1 (mos10)55, tpr2 (SALK_112730)56, tpr3 (WiscDsLox350C06)56, tpr4 (SALK_150008)55 and tpl tpr1 tpr455 mutants are in the Col-0 background. The tpl-1 (point mutation, N174H)21 mutants are in the Ler background. The transgenic lines pTPL::TPL53 and pREV::REV-GR-HA57 are in the Col-0 background. The pREV::REV-Venus20, pSTM::STM-Venus20 and p35S::STM-GR58 lines are in the Ler background. The mir394b-1 was kindly provided by Xuemei Chen (Peking University). The tpl-1 and pTPL::TPL were kindly provided by Lei Wang (Institute of Botany, CAS). The tpl, tpr1, tpr2, tpr3, tpr4 and tpl tpr1 tpr4 were kindly provided by Jingbo Jin (Institute of Botany, CAS).
In this study, lcr-1 (SALK_136833) mutants were obtained from the AraShare Arabidopsis Stock Center. The mir394a-cr, mir394b-cr and lcr-cr mutants were generated via a CRISPR-Cas9 system in the Col-0 background. The mir394a/b double mutant was obtained via genetic crosses of mir394a-cr and mir394b-1. The mir394a/b lcr-1 triple mutant was generated via a genetic cross of mir394a/b and lcr-1. The pREV::REV-Venus p35S::LCR4m, pSTM::STM-Venus p35S::LCR4m, pREV::REV-GR-HA p35S::LCR4m, p35S::STM-GR p35S::LCR4m and p35S::LCR4m tpl-1 were generated via genetic crosses. The p35S::MIR394A, p35S::MIR394B, p35S::LCR, p35S::LCR4m, p35S::LCR5m, p35S::LCRF-box, p35S::LCR4mΔF-box, pLCR::LCR, pLCR::LCR4m, pLCR::LCRΔF-box, pLCR::LCR4mΔF-box, pMIR394A::GFPer, pMIR394B::GFPer, gLCR::GFP, pREV::LCR5m, pKAN1::LCR5m and pTPL::GFP transgenic lines were generated in the Col-0 background. The p35S::TPL-GFP transgenic lines were individually generated in the Col-0 and lcr-1 backgrounds. The p35S::TPL-mCherry transgenic lines were individually generated in the Col-0, p35S::LCRΔF-box and p35S::LCR4m backgrounds. All the transgenic lines used in this work were generated via the Agrobacterium-mediated GV3101 transformation method. For selection of transgenic plants, seeds were sterilized and germinated on MS medium supplemented with 25 mg/L hygromycin or 50 mg/L kanamycin. The mentioned material genotyping primers are listed in Supplementary Table 1.
Vector construction
To construct the mir394a-cr, mir394b-cr and lcr-cr mutants, the sgRNA sequence targeting pre-miR394a, pre-miR394b or LCR was synthesized and inserted into the pBUE401 CRISPR/Cas9 vector using the BsaI enzyme (NEB)59. To construct the expression vector, MIR394A (3000 bp promoter) and MIR394B (3000 bp promoter) were amplified from Col-0 genomic DNA and inserted into the TSK108 vector, and subsequently, constructed into the pMDC204 vector via gateway. The genomics-LCR (2281 bp promoter and the first exon, intron, and three amino acids of the second exon) and promoter-TPL (2548 bp promoter) were amplified from Col-0 genomic DNA and inserted into the pCambia1300 vector via homologous recombination constructs. To construct the LCR overexpression vector, the LCR (the first exon, intron, and the second exon) was amplified from Col-0 genomic DNA. The LCR4m and LCR5m were constructed by point mutations39,43. The LCRΔF-box and LCR4mΔF-box were amplified from two fragments via the corresponding primers to remove the F-box region. The PCR fragments were subsequently cloned and inserted into the pGEM-T Easy vector (Promega) and individually transferred into the LCR promoter and 35S promoter-driven expression vector pCambia1300 via digestion and ligation. To construct the LCR ectopic expression vector, REV (3000 bp promoter) and KAN1 (3000 bp promoter) were amplified from Col-0 genomic DNA and inserted upstream of the LCR5m coding sequence (CDS). The fragment was subsequently introduced into the express vector pCambia1300. To construct the TPL overexpression vector, the CDS of TPL was amplified from Col-0 cDNA and individually introduced into the expression vectors pCambia2300 and pCambia1300 driven by the 35S promoter. The primers used for vector construction are listed in Supplementary Table 1.
Total RNA extraction, qRT-PCR analysis and northern blotting
Total RNA was extracted from 30-day-old vegetative shoot apices via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Thermo Fisher). First-strand cDNA was synthesized with 1 ng of total RNA via oligo-dT primers and the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. qRT-PCR was performed using Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) according to the manufacturer’s instructions on a Bio-Rad CFX96 real-time PCR detection system with a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Beijing, China). ACTIN2 was used to normalize the relative expression. The qRT-PCR primers (Supplementary Table 1) were used to amplify each gene. The mRNA expression levels were calculated via the 2−∆∆Ct method. The data were repeated with three biological and at least two technical replicates.
For small RNA northern blotting60. Total RNA (10 μg) was resolved on 15% urea-PAGE gels and then transferred to a nylon membrane for 1 h at 300 mA. The nylon membrane was subsequently hybridized overnight at 50 °C with 3’ biotin-marked DNA probes to detect miRNAs. The probes used are listed in Supplementary Table 1.
RNA-seq and sRNA-seq
For RNA-seq, vegetative shoot apex materials (grown under short-day conditions for 30 days) were collected from mir394b-1, mir394a-cr, pLCR::LCR4m, miR394A-OX, p35S::LCR4m, tpl tpr1 tpr4 and WT individually for RNA extraction. An RNA-seq libraries were constructed via standard Illumina protocols. Deep sequencing was performed via an Illumina NovaSeq 6000 platform in 150-nucleotide paired-end mode by Berry Genomics (Beijing). Two biological replicates were prepared and analyzed for each sample. Each replicate was obtained by pooling samples from at least fifty plants. The raw sequencing reads were quality trimmed via Trim Galore (v0.6.5), and the clean reads were mapped to the Arabidopsis thaliana reference genome sequence (TAIR10 Genome Release) via HISTA2 (v2.2.1)61. The count files were then used as input for DEG analysis via DESeq2 (v1.36.0)62 with the threshold of Log2 (fold change) >1 and an FDR < 0.05 were set as the cut off criteria for significant differences. The raw counts were further normalized to fragments per kilobase of transcript per million fragments mapped (FPKM). Gene co-expression models of all DEGs were constructed using the k-means method by Z-scaled FPKM in R (v4.0.2), and the cluster number was determined by the figure of merit approach63. The gene expression heat map was displayed using the R package ComplexHeatmap (v2.4.3)64. Functional enrichment was performed via the R package clusterProfiler (v4.0)65, and GO annotation files were generated from TAIR Annotation. Correlation coefficients represent the distance between any two samples, and data were conducted by Cor function in R (v4.0.2)66.
For sRNA-seq, the seedlings of Col-0 grown under short-day conditions for 10 days and vegetative shoot apex of Col-0 grown under short-day conditions for 30 days were collected with two biological replicates, individually. The sRNA-seq libraries were prepared from 1 μg of total RNA via standard Illumina protocols. Deep sequencing was performed with insertions between 18 and 40 bp on an Illumina NextSeq 500 by Berry Genomics (Beijing). The raw sRNA-seq data were trimmed via Cutadapt (v1.12)67 to remove adapters and low-quality reads. Reads that were 18–30 nt in length were retained. The retained reads were annotated via the Infernal package (v1.1.2) in Rfam (https://www.sanger.ac.uk/tool/rfam/). The number of reads mapped to known miRNA sequences was determined via FeatureCounts (v2.0.0)68 on the basis of the Arabidopsis miRNA models in miRBase (release 22.1). The transcription levels of the miRNAs were estimated via the R package edgeR69 on the basis of the read counts, which were normalized to CPM (read counts per million mapped reads) values. DE miRNAs were identified via DEseq2 (v1.36.0)62 with the threshold of Log2 (fold change) >1 and an FDR < 0.05.
Observation of the AM phenotype
For visualization of AMs, plants were placed under a stereoscope, and observed whether buds had formed in the leaf axils of rosette and cauline leaves in order from the oldest to the youngest leaf axils. Among them, the cotyledons did not form AM, so the cotyledons were removed during the statistical analysis. We performed at least three independent biological experiments, and more than 20 individual plants were counted each time.
Live imaging
For live imaging10,70, leaves between P8 and P11 were detached from seedlings using dissecting medium (3% agarose) and laid flat on imaging medium (1/2 MS medium with 1% agarose on the top). FM4-64 (Thermo Fisher) was applied to the meristem for 20 min for imaging. For time-lapse live imaging, the leaves were transferred back to growth medium (1/2 MS medium with 2% sucrose, 0.0005% (w/w) folic acid, 0.01% (w/w) myo-inositol, pH 6.0 and 0.3% phytagel) under normal growth conditions after each image. All live imaging experiments were performed via the water dipping lens of an Olympus (FV1000MPE) confocal microscope.
Agarose sectioning
For agarose sectioning, shoot apex were collected by removing the leaves and then immediately placed in 2.5% paraformaldehyde (PFA; Sigma-Aldrich) at pH 7.0. After vacuum infiltration for 30 min on ice, the samples were stored overnight at 4 °C. Fixed tissue samples were washed with 10% sucrose (prepared with 1% PFA at pH 7.0) for 20 min, 20% sucrose (prepared with 1% PFA at pH 7.0) for 20 min, and 30% sucrose (prepared with 1% PFA at pH 7.0) for 30 min, successively. The samples were then embedded in 6% low-melting-point agarose (Promega) liquid gel at 37 °C and placed at 4 °C for 30 min to solidify. Sections (40 μm) were prepared using a Leica VT1000S vibratome.
TBO staining and RNA in situ hybridization
For TBO staining71 and RNA in situ hybridization72,73, the shoot apex were harvested and fixed with 4% paraformaldehyde. After being embedded in wax, the sections were sliced into 8 μm sections using a Leica instrument (RM2255). The miR394 probes were synthesized via LNA modification, and 3’ digoxigenin-labeled mRNA probes were amplified from Col-0 cDNA with gene-specific primers containing the T7 promoter sequence at the 5’ end and synthesized using a DIG Northern Starter Kit (Roche, 11277073910). The primers used for probe synthesis are detailed in Supplementary Table 1.
Subcellular localization
For colocalization of LCR and TPL in Arabidopsis protoplasts, the CDS of LCR was amplified and ligated into pBI221-GFP, and TPL was amplified and ligated into the pBI221-mCherry vector. All primers used are listed in Supplementary Table 1. The plasmids were subsequently transformed into protoplasts obtained from the leaf sheaths of 21-day-old seedlings via the polyethylene glycol-mediated transient expression system. The transformed protoplasts were observed via confocal microscopy.
BiFC assays
The full-length CDS of the TPL sequence was recombined into pSPYNE, while the CDS of the LCR and LCRΔF-box sequences were recombined into pSPYCE. All primers used for BiFC are listed in Supplementary Table 1. Each construct was subsequently introduced into Agrobacterium GV3101. Then, different combinations were co-infiltrated into 4-week-old N. benthamiana leaves as indicated for transient expression. H2B-mCherry was used as a nuclear marker. After co-transformation for 36–48 h, the YFP signals were analyzed via confocal microscopy.
Confocal microscopy, optical microscopy, and scanning electron microscopy
Confocal microscopy images were taken with a Zeiss (LSM 980) or an Olympus (FV1000 MPE) microscope. The excitation and detection wavelengths were 488 nm and 505–525 nm for GFP; 488 nm and 650–700 nm for autofluorescence; 514 nm and 580–620 nm for FM4-64, 514 nm and 524–550 nm for Venus; and 561 nm and 585–640 nm for mCherry. Optical photographs were taken with an Olympus (BX53) microscope equipped with a Canon camera. Scanning electron microscopy was performed by using a Hitachi S-4800 field-emission variable pressure scanning electron microscope after standard tissue preparation for cellular observation. We observed at least three independent transgenic lines in each genetic background and confirmed that the patterns were consistent within each genotype.
Chemical treatments
For Dex treatment, a 10 μM Dex (Sigma-Aldrich) solution containing 0.01% (v/v) Silwet-77 was applied to vegetative shoot apex tissues every two days. After 30 days of continuous treatment, the plants were transferred to long-day conditions for 30 days, and the AM initiation phenotype was observed.
For MG132 treatment, equal numbers of seedlings were cut into 1–2 mm slices and treated with 50 μM MG132 (Sigma-Aldrich) solution or without MG132 (0.1% DMSO) for 5 h (exposed to light) or 24 h (exposed to the dark). Samples were collected at 5, 12 and 24 h after treatment for protein extraction and detected by immunoblotting.
Protein extraction and immunoprecipitation
For total protein extraction, seedlings were harvested and ground in liquid nitrogen, and the powder was resuspended in IP buffer (50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.5% NP-40, 1 mM PMSF, 2 mM NaF, 50 μM MG132 and 1x proteinase inhibitor cocktail (Roche)) and incubated for 30 min with gentle rotation at 4 °C. The protein suspensions were centrifuged at 15,000 × g for 10 min (repeated once) to remove debris, and the supernatant was collected for further experiments.
For IP-MS, the same protein extraction procedure was individually performed for 2 g of p35S::LCR-GFP, p35S::LCR4m-GFP and p35S::GFP seedling material. The supernatant was supplemented with precleared GFP Trap beads (ChromoTek) and incubated at 4 °C with gentle agitation for 2 h. The beads were washed at least five times with washing buffer-1 (50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 150 mM NaCl, 0.5% NP-40, 1 mM DTT, 1 mM PMSF and 50 μM MG132) and washing buffer-2 (50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA (pH 8.0), 150 mM NaCl, 1 mM PMSF and 50 μM MG132). Finally, the protein-bead complex was separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After Coomassie blue staining, the target gel lanes were cut and sliced, and digestion was carried out with trypsin. After staining and trypsin digestion, the polypeptide was subjected to mass spectrometry via an Orbitrap Fusion Lumos instrument (Thermo Fisher Scientific) from the CAS Centre of the Institute of Botany, Key Laboratory of Plant Molecular Physiology. The search engine was used for protein identification by searching against the UniProt protein database (https://www.uniprot.org/). The false discovery rate (FDR) for protein identification was also set at 0.01. The significance threshold was set at p < 0.05.
For Co-IP, the WT, tpl tpr1 tpr4 and p35S::LCR4m-GFP supernatants were incubated with GFP-Trap for 2 h at 4 °C, after which the beads were washed with washing buffer-1 and washing buffer-2 three times. The protein was eluted from the beads in 2XSDS sample buffer (120 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.04% bromophenol blue, and 10% β-mercaptoethanol) by heating at 95 °C for 5 min. The proteins were subsequently transferred to 8% SDS-PAGE gels and detected with anti-GFP and anti-TPL antibodies.
For in vivo ubiquitination assays, the p35S::TPL-GFP and p35S::TPL-GFP lcr-1 protein supernatants were incubated with GFP-Traps for 2 h at 4 °C and detected with anti-GFP and anti-ubiquitin antibodies.
The primary antibodies used in this study included anti-GFP (598, MBL, 1:1000 dilution), anti-mCherry (PM005, MBL, 1:1000 dilution), anti-ubiquitin (Ab134953, Abcam, 1:1000 dilution), anti-TPL (A21285, ABclonal, 1:1000 dilution) and anti-ACTIN (AC009, ABclonal, 1:3000 dilution) antibodies. The expression of protein was counted using ImageJ (National Institutes of Health, USA) per sample.
Y2H assays
LCR, TPL, TPR1, TPR2, TPR3 and TPR4 were amplified from Col-0 cDNA and subsequently cloned and inserted into the prey vector pGADT7 (630442, Clontech) or bait vector pGBKT7 (630443, Clontech). The primers are listed in Supplementary Table 1. Standard yeast extract-peptone-dextrose plus adenine (YPAD) media were used, and a lithium acetate protocol was used to transform the digested plasmids. The yeast strain AH109 was co-transformed with various bait and prey construct combinations via heat shock treatment (42 °C, 15 min). Equal numbers of co-transformed yeast cells were grown on SD-Leu/-Trp medium for 3 days (30 °C). The relevant yeast cells were serially diluted (1:10, 1:100, 1:1000) and added to SD-Leu/-Trp/-His/-Ade solid selective medium containing X-α-gal to detect protein interactions.
Statistics and reproducibility
Statistical analyses to examine significant differences and result visualization were performed with the GraphPad Prism 9 software. Significance was determined by p < 0.05 [*] or p < 0.01 [**]. All gel blots/plots were performed at least three times.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The raw data of regular RNA-Seq and small RNA-Seq in this study have been deposited in National Centre for Biotechnology Information (NCBI) (accession numbers PRJNA1101744 and PRJNA1164152). All plasmids and strains supporting the finding of this study are available from the corresponding author upon request. Source data are provided with this paper.
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Acknowledgements
We would like to thank Prof. Xuemei Chen (Peking University) for miR394 genetic materials, helpful discussions and critical reading for the manuscript. We would like to thank Prof. Lei Wang (Institute of Botany) for the seeds of tpl-1 and pTPL::TPL, and Prof. Jingbo Jin (Institute of Botany) for the seeds of tpl, tpr1, tpr2, tpr3, tpr4 and tpl tpr1 tpr4 mutants. We also thank Dr. Fengqin Dong, Dr. Jingquan Li, Dr. Xiuping Xu and Dr. Zhuang Lu (Institute of Botany) for technical assistance. This work was supported by the National Key Research and Development Program of China (2023YFF1001301), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDA24010106-2) to C.Z.
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C.Z. and L.L. designed the research. L.L. performed most of the experiments and analyzed the data. B.H. analyzed the RNA seq and sRNA seq data. S.G. performed the northern blot experiments. Z.X. and T.W. conducted some of the experiments and provided suggestions for RNA in situ hybridization and protein experiments. C.Z. and L.L. wrote the manuscript.
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Liu, L., Hu, B., Guo, S. et al. miR394 and LCR cooperate with TPL to regulate AM initiation. Nat Commun 15, 10156 (2024). https://doi.org/10.1038/s41467-024-54494-6
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DOI: https://doi.org/10.1038/s41467-024-54494-6
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