miR394 and LCR cooperate with TPL to regulate AM initiation

doi:10.21203/rs.3.rs-4268061/v1

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miR394 and LCR cooperate with TPL to regulate AM initiation

https://doi.org/10.21203/rs.3.rs-4268061/v1

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published 23 Nov, 2024

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The plant architecture is a main determinate of crop yield, and lateral branching significantly influences the number of inflorescences and seeds. Axillary buds support lateral branch growth, and the development of axillary buds includes two stages, initiation and outgrowth. Extensive studies on the outgrowth stage have uncovered fine regulatory mechanisms of branch growth, while our understanding of axillary bud initiation remains unclear. This work aims to study how miRNA regulate axillary bud initiation. By constructing small RNA library and screening mutant population, we identified miR394s promote axillary bud initiation. We found that the initiation of axillary buds is specifically induced by miR394 and repressed by its target LEAF CURLING RESPONSIVENESS (LCR) in the center of leaf axils. Using promoter-driven fluorescent tags and in situ hybridization, we showed that miR394 localized in the position where AMs initiate. Through molecular and genetic research, we found miR394 may regulate REVOLUTA-STM pathway to establish axillary meristem. Immunoprecipitation and mass spectrometry studies showed that LCR, as an F-box protein, interacted with TOPLESS (TPL) proteins and participate in ubiquitinated protein degradation. Our results reveal an important mechanism that miR394s regulated LCR accelerates the degradation of TPL to precisely modulate axillary bud initiation.

Biological sciences/Plant sciences/Plant development/Plant morphogenesis

Biological sciences/Genetics/Epigenetics/Gene silencing

axillary meristem

miR394

LCR

TPL

Arabidopsis thaliana

The axillary meristem (AM) is a highly dynamic and active stem cell system which located in leaf axils and responsible for the generation of lateral shoots in plants^1,2. AM development includes initiation and bud outgrowth³. During AM initiation, a morphologically detectable bump is formed in the leaf axil and develops into a bud^4,5,6. Much less is known about AM initiation due to its subtle structure. Until now, several transcription factors are featured to regulate AM initiation^{5,7,8,9,10,11,12,13,14,15}.

As AMs initiate from the boundary regions at the base of adaxial side of leaves, some transcription factors, including CUP SHAPED COTYLEDON (CUCs)⁹, LATERAL SUPPRESSOR (LAS)⁸ and REGULATOR OF AXILLARY MERISTEMS (RAXs)^14,16 enrich in the boundary and specify boundary formation before AM initiation. The AM initiation requires a meristematic cell population continuously expressing marker gene SHOOT MERISTEMLESS (STM)^5,10,15. The expression of STM is dynamic and is 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 was highly expressed in leaf axils older than P₁₀, which promotes the formation of AMs¹⁰. After the induction of STM and gain of meristem activity, homeodomain transcription factor WUSCHEL (WUS) promotes AM initiation in the leaf axils around the P₁₃ leaves¹². WUS expression is also feedback regulated by CLAVATA3 (CLV3) in the AMs¹³. REVOLUTA (REV) which belongs to the HD-ZIPIII family targeted by miR165/166 expresses coordinately with AM initiation and is the only member of this family to activate stem cells during AM initiation^4,17,18. REV expresses in a low level on the adaxial side of young leaf axils (< P₉) and increases in the old leaf axils (> P₁₀) when AMs initiate^15,19. REV was found to promote STM directly by binding STM promoter to activate AM initiation¹⁰. Auxin and cytokine are two major hormones regulating meristem development^20,21. In boundary and AMs, auxin and cytokine as well as brassinosteroids (BRs) also play vital roles^12,19,22,23. In some cases, transcription factors in these hormonal pathways combined with general transcription repressor, such as TPL to precisely regulate meristem marker genes^24,25,26,27.

MicroRNAs (miRNAs), endogenous small noncoding RNAs with a length ranging between 20 and 24 nucleotides, play critical roles in plant development^{28,29,30,31,32,33,34}. miRNAs control target gene expression at the post-transcriptional level by directly cleaving or inhibiting translation^35,36. However, it is still unclear how miRNAs participate in the development of AMs. Although miR165/166 as a cleaver of REV and the role of miRNA effector ARGONAUTE10 (AGO10) were reported in AM initiation^19,37,38, whether other miRNAs participate in AM development and how they function are unknown.

miR394 is a highly conserved miRNA of 20 nucleotides, and first identified in Arabidopsis thaliana^39,40. Previous studies have reported that miR394 is involved in regulating meristem development, leaf morphogenesis, and stress resistance pathways^{41,42,43,44,45,46,47,48,49}. In the shoot meristem of Arabidopsis thaliana, miR394 acts as a mobile signal from the protoderm on the surface of the SAM, maintaining stem cell activity and affecting the expression of CLAVTATA3 (CLV3), a SAM-specific gene. In the SAM organizing center (OC), miR394b inhibits its direct target Leaf Curling Responsiveness (LCR) transcript levels⁴³. Furthermore, LCR may associate with the MLP-LIKE PROTEIN 28 (MLP28) family member to regulate SAM development⁴⁵. However, the involvement of miR394 and LCR in AM development has not been reported and the pathways mediated by miR394 and the F-box proteins remain enigmatic.

In this study, we constructed small RNA library used boundary enrichment tissues and screened miRNAs potentially involved in AM development, and discovered that miR394 was highly expressed in AMs. We found that miR394 control LCR expression to establish stem cell niches de novo during AM initiation in Arabidopsis. miR394/LCR expression exhibited temporal and spatial patterns that finely matches the dynamic of AM initiation: miR394/LCR were enriched in the adjacent domain between leaf adaxial side and boundary, where AMs initiate. The expression was continued on the AMs throughout AM development and remained in the buds. In addition, we found LCR regulated STM and REV expression downstream. Further, LCR was demonstrated to act with a specific Skp1/Cullin1/F-box (SCF) E3 ligase complex and target TOPLESS (TPL), which functions as a positive regulator of AM initiation. Therefore, our findings identify three new modulators of AM initiation and establish a novel pathway mediated by miR394s and F-box protein regulating AMs.

MiR394s promoted and LCR inhibited AM initiation in Arabidopsis

In order to identify miRNAs involved in axillary meristem formation, we performed small RNA-seq with boundary enrichment tissues (shoot apices without leaves) to assess global changes of miRNAs in leaf axils. Significant higher expressions of miR394a and miR394b were found in shoot apices by compared with that in 10 days old leaves (Fig. 1a). Previous reports showed, mir394b-1 in the ago10-1 background exhibited a “pin-like” shoot indicative of stem cell defects ^43,50. Consistently, in Arabidopsis, by screening small RNA related mutant population, we identified that AMs in rosette leaves of mir394b-1 failed to initiate, but could form normally in the Col-0 (Fig. 1b-c). To understand in more detail how miR394s regulate the formation of AMs in rosette leaf axils in the vegetative shoots. We first knocked out mir394a and miR394b by the CRISPR/Cas9 genome editing approach in mir394a-cr and mir394b-cr lines and generated overexpression lines miR394A-OX and miR394B-OX (Supplementary Fig. 1a-f). The mutant mir394a-cr showed AM defects as mir394b-1 (Fig. 1b-c). We further quantified AMs of mir394a/b double mutants and observed a significantly decrease of AMs compared to each single mutant (Fig. 1b-c), suggesting that miR394a and miR394b may act redundantly in controlling AM initiation. In contrast, overexpression of miR394 did not result in a change of AM number compared with WT (Supplementary Fig. 6). To explain miR394 functions in AM initiation, we considered whether the LCR genes which were known as miR394 targets are the cause for the defects of AMs³⁹. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis showed that, the expression levels of pri-MIR394a and pri-MIR394b were extremely low in mir394a/b, while LCR were detected with a much higher expression under the same experimental conditions (Supplementary Fig. 1e). To analyze whether local LCR expression in the leaf axils inhibits AM initiation, we obtained a loss-of-function mutant of LCR with T-DNA insertion, lcr-1 (SaIL-136833) and generated LCR CRISPR mutants lcr-cr by designing sgRNAs to target LCR (Supplementary Fig. 2a). After PCR confirmation, we obtained three independent homozygous lines of lcr-cr (Supplementary Fig. 2). Both lcr-cr (#1, #2 and #3) and lcr-1 mutants showed dramatically reduced LCR mRNA levels (Supplementary Fig. 2b-d). Meanwhile, we expressed LCR and miR394-resistant versions LCR-4m⁴³ and LCR-5m⁴⁷ in Col-0 plants, driven by the LCR promoter or the CaMV 35S promoter (Supplementary Fig. 4a). After genotyping, combined with qRT-PCR and western blot analysis, we obtained three independent homozygous lines of LCR-OX and LCR4m-OX (Supplementary Fig. 4b-e). As expected for miR394 targets, LCR4m mRNA levels were highly accumulated in pLCR::LCR4m and p35S::LCR4m (Supplementary Fig. 4c) and led to a strong phenotype of AM deficiency (Fig. 1b, Supplementary Fig. 5a-l). In contrast to the overexpression plants, LCR loss-of-function mutants grew faster and formed normal AMs in rosette leaves (Supplementary Fig. 6a). Notably, the AM initiation defect of mir394a/b could be rescued by lcr-1 (Supplementary Fig. 6a) confirming that miR394s promote AM initiation through downregulation of LCR mRNA levels. We observed AM morphology of the mir394a/b double mutant, pLCR::LCR4m, p35S::LCR4m and WT using scanning electron microscopy. While wild-type plants had obvious lateral buds in axils of mature rosette leaves, mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m lacked lateral buds (Fig. 1c). We detected anatomical changes at the leaf axils through tissue sections. In the WT leaf axils of P₁₄ to P₁₆ stage, more densely stained cells form morphologically distinguishable bumps. In mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m, the frequency of densely stained cells and AM structures were highly reduced in the leaf axils at the same stages (Fig. 1d). WT plants had an obvious, dome-shaped AM in the axil of P₁₅. While, mir394a/b double mutant, pLCR::LCR4m and p35S::LCR4m plants did not show an AM bulge at the same stage (Fig. 1d). These results suggested that inhibition of LCR expression by miR394 is necessary for AM initiation in Arabidopsis. LCR encodes a conserved F-box protein^51,52,53 (Supplementary Fig. 3a-d). To confirm whether LCR affects AM initiation through F-box domain, we constructed overexpression lines with F-box domain removed so that the target proteins could not be degraded by the mutated version LCR^△F-box (Supplementary Fig. 4a-e). We tracked changes at the leaf axils of LCR^△F-box-OX and LCR4m^△F-box-OX plants, and found normal AMs were formed (Supplementary Fig. 6b). These results indicated that LCR may act as a negative regulator in AM initiation through its F-box domain.

miR394 and LCR expressed on the adaxial side of leaves and AMs.

AMs are initiated at the axils of subtending leaves in Arabidopsis thaliana during early shoot development. The AM initiation is regulated temporally and spatially and formed morphologically distinguishable structure by P₁₁. We therefore tested whether miR394 exists in shoot axils. To visualize the real-time dynamics of miR394 expression, we constructed the pMIR394A::GFPer and pMIR394B::GFPer reporter lines (Supplementary Fig. 7a-b) and tracked miR394s transcription in young leaf axils. We found that MIR394A had a strong signal, in the boundary region of emerging leaf primordia and SAM, and this signal spread into the adaxial domain of the leaves as well but decreased in AMs (Fig. 2a and Supplementary Fig. 8a). In addition, a similar pattern of MIR394B expression was detected in primordia and boundary cells as MIR394A but the signals in AMs were relatively higher than MIR394A (Fig. 2b and Supplementary Fig. 8b). RNA in situ hybridization assays revealed that mature miR394s accumulated in the SAM, AMs, vasculature, and adaxial side of leaves (Fig. 2c and Supplementary Fig. 9a-p). These results indicated that miR394a and miR394b were co-expressed in the boundary, but distinguished and partially overlapped in AMs implying the overlapped and respective roles of the family members. To explore the expression patterns of LCR, the promoter of LCR with the first exon, three amino acids of the second exon were amplified to fuse with GFP and transformed into the Col-0 ecotype of Arabidopsis (Supplementary Fig. 7a-b). In T3 seedlings, gLCR::GFP expression was examined in the shoot apex. Similar to miR394, LCR was also expressed in adaxial domain of the leaves, and strongly expressed in boundary, SAM and AMs (Fig. 2d and Supplementary Fig. 8c). In situ hybridization using pLCR::LCR4m plants revealed the accumulation of the LCR transcript in leaf axils as the pattern observed in gLCR::GFP (Fig. 2e). To further confirm the importance of LCR in adaxial domain, we specifically expressed LCR4m, 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 forming a phenotype similar to pLCR::LCR4m, with severe defects of AMs, but pKAN1::LCR4m remains the same as the wild-type (Supplementary Fig. 10a-c). The temporal and spatial patterns of miR394 and LCR expression coincided perfectly with those of AM initiation. Transgenic plants harboring the p35S::LCR-GFP and p35S::LCR4m-GFP were obtained and GFP signals were verified in root (Supplementary Fig. 11a,e). However, the signal of LCR4m was detected in the shoot apex but LCR was not (Supplementary Fig. 11b-d and f-h). Moreover, the western blot results also confirmed that LCR4m protein was significantly higher expressed than LCR (Supplementary Fig. 11m), corroborating the expression of LCR was strictly controlled by miR394s in shoot apex. In addition, after removing the F-box domain, LCR^△F−box-GFP protein still showed fluorescent signals in nucleus (Supplementary Fig. 11i), but a weak LCR^△F−box-GFP signal appeared in adaxial domain of leaves (Supplementary Fig. 11j-l), and the level of LCR^△F−box protein was also increased (Supplementary Fig. 11m). By treating with MG132, we also found the degradation rate of LCR4m was slowed down (Supplementary Fig. 11n). These results suggested that LCR degraded target proteins and itself through 26s proteasome in shoot apex.

miR394s activated REV and STM pathway during AM initiation

To investigate the targets of miR394s and the pathways mediated by miR394/LCR during AM initiation, transcriptomic analysis using boundary enrichment tissues of miR394 mutants and OE lines were performed. We identified 2224 differentially expressed genes (DEGs) in MIR394A-OX lines, 3002 DEGs in miR394a-cr mutants, 2322 DEGs in miR394b-1 mutants and 2539 DEGs in LCR4m-OX lines, individually (Supplementary Fig. 12a). GO analysis extracted several pathways shared in these plants in which post-embryo and leaf developmental pathways focused in miR394s mutants and LCR4m-OX lines (Supplementary Fig. 12b). Cluster analysis indicated that LCR4m-OX displayed a more similar profile to miR394b-1 mutant compared with miR394a-cr (Supplementary Fig. 12c). Meristem marker genes were listed in the transcriptomic analysis. In LCR4m OX lines with the most severe phenotype of AMs, REV were dramatically decreased compared with other genes. However, in other lines, the marker genes did not show significant trend as previous report about miR394b-1 mutant that only in ago10 background the mutant displayed obvious phenotype and the changes in meristem markers were able to detect⁴³ (Supplementary Fig. 12d). Next, we used RT-qPCR and confirmed the upregulation of STM and REV in LCR4m OX lines. In consistent, mir394a/b mutants and LCR4m-OX plants showed a significant decrease of STM and REV in the axil enriched tissues with leaves removal compared with WT (Supplementary FigS12e). Other marker genes were also detected, CUC3, LAS, RAX1, CLV3 decreased while KAN3 increased in LCR4m-OX plants. However, the change of WUS was not significant in the axil enriched tissues (Supplementary FigS12e). Small RNA library analysis showed a few miRNAs changes in miR394s mutants and MIR394A-OX lines. miR391 increased and miR169f decreased in miR394a-cr mutant while their predicted targets showed opposite expression patterns (Supplementary FigS13a,c). miR167 was found to be significantly down-regulated in MIR394A-OX lines, and its target gene ARF8 (AT5G37020) also had an opposite expression pattern (Supplementary FigS13b-c).

We crossed the marker lines pSTM::STM-Venus⁵⁴ and pREV::REV-Venus²³ to the p35S::LCR4m transgenic lines. In early leaf axils (< P₉), the expression of STM persisted in WT (Fig. 3a). STM remained expression in p35S::LCR4m, but was much lower than WT (Fig. 3b). Starting from P₉, the expression of STM in leaf axils of p35S::LCR4m was restricted and even completely disappeared in P₁₃ (Fig. 3b) while STM was activated in WT. The low expression of STM may explain the deficiency of AM initiation in LCR4m-OX plants. We next asked whether REV expression is affected in the p35S::LCR4m plants. In the WT, REV-Venus signals were present in AMs from the P₉ (Fig. 3c). However, during leaf maturation, REV nearly undetectable in AMs and adaxial side of leaves in the p35S::LCR4m plants (Fig. 3d). To monitor the AM initiation quantitatively, we performed live imaging of leaf axils that were isolated from the shoot apex under short-day conditions (Supplementary Fig. 14a-h). We observed no axillary bud formation in detached axils of LCR4m-OX from P_12/13 and performed time-lapse live imaging with detached P₁₃ axil to closely trace STM and REV expression and AM initiation. By cultivating, no cells in the LCR4m-OX background maintained STM and REV expression, while the number of STM and REV expressing AM progenitor cells had dramatically increased in the WT. To determine whether the loss of STM and REV expression in leaf axil was responsible for the AM initiation defects in LCR4m-OX, we introduced inducible STM-GR (glucocorticoid receptor) under the 35S promoter or the REV-GR (glucocorticoid receptor) under the REV promoter. We performed dexamethasone (DEX) induction on LCR4m-OX transgenic plants, it was found that both transgenic lines were able to significantly restore the formation of AMs (Supplementary FigS14i-j). These observations confirmed that the inhibition of LCR by miR394 is necessary for promoting STM and REV expression in AM initiation.

LCR associated with TPL in leaf axils and AMs

To identify potential substrates of LCR, we searched for its interactome by Immunoprecipitation-Mass Spectrometry (IP-MS). Boundary enrichment tissues of entrained p35S::LCR-GFP and p35S::LCR4m-GFP transgenic seedlings were collected to do immunoprecipitation (Fig. 4a). In total, we got 460 proteins immunoprecipitated by anti-GFP anti-body among two biological replicates and selected seven proteins to verify. 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 maintenance^24,55. We thus tested whether LCR can interact with TPL. As expected, Y2H showed that LCR proteins displayed stronger interaction with TPL (Fig. 4b). Domain deletion analysis revealed that C-terminus of LCR (164–467 aa) could interact with TPL (Fig. 4b). Moreover, truncated TPL containing aa 93 to 352 can interact with LCR (Supplemental Fig. 15a-b). The Arabidopsis genome contains five TPL family members, namely TPL, TPR1 (TOPLESS-RELATED 1), TPR2 (TOPLESS-RELATED 2), TPR3 (TOPLESS-RELATED 3) and TPR4 (TOPLESS-RELATED 4)²⁴. Next, we characterized the potential physical interaction between LCR and the TPL homologs TPR1, TPR2, TPR3, and TPR4 by Y2H (Supplemental Fig. 15c-d). The results revealed that LCR could interact with TPR1, TPR2 and TPR4. To further confirm LCR directly associating with the TPL protein, we performed bimolecular fluorescence complementation (BiFC) experiments in tobacco leaves and observed an interaction between LCR and TPL in vivo (Fig. 4c).

To investigate the acts of LCR on TPL and whether the expression of TPL were altered by LCR. We first did qRT-PCR analysis and the results demonstrated that TPL and TPR1 were slightly down-regulated and TPR3 slightly up-regulated in pLCR::LCR4m, but the expressions of TPR2 and TPR4 were not significantly affected by pLCR::LCR4m compared with the WT (Supplemental Fig. 16d). Notably, in protein level, TPL and LCR were co-localized in the nucleus (Supplemental Fig. 16e). To quantify the levels of TPL family proteins, we used antibody made by TPL conserved domains to detect all the family members. We found that the protein level in tpl was lower than WT and were substantially reduced in tpl tpr1 tpr4. The protein level in lcr-1 was slightly increased, while was decreased in pLCR::LCR4m (Supplemental Fig. 16g). We also tested TPL accumulation in p35S::LCR^△F-box and p35S::LCR4m. Immunoblot analysis showed that TPL was significantly reduced in the p35S::LCR4m but not changed in p35S::LCR^△F-box (Supplemental Fig. 16f) indicating that F-box of LCR was important for TPL degradation .

To test whether TPL can be ubiquitinated by LCR, we extracted the total proteins from p35S::TPL-GFP in WT or lcr-1 mutant background respectively and performed immunoprecipitation with anti-GFP antibody. Subsequent immunoblot analysis of the IP proteins using anti-GFP and anti-Ubi antibodies revealed that TPL was ubiquitinated in planta. The ubiquitination of TPL was decreased in lcr-1 compared with WT (Supplemental Fig. 16h). To confirm whether the degradation of TPL was mediated by the 26S proteasome, we used MG132 to treat plants. The presence of MG132 stabilized TPL, which displayed a slower degradation time course compared with the absence of MG132 (Fig. 4d). These results indicated that TPL was a substrate with ubiquitin modification of LCR.

TPL expressed in leaf axils and promoted AM initiation

LCR and TPL proteins physically interacted with each other and LCR may negatively regulated TPL abundance by protein degradation. Therefore, we wanted to know whether TPL regulates AM initiation. We comprehensively observed AM phenotype of TPL family mutants such as tpl-1, tpl, tpr1, tpr2, tpr3, tpr4 and tpl tpr1 tpr4 (Fig. 5a and Supplementary Fig. 17a-b). In tpl-1 plants, the number of leaves was significantly reduced, and AMs were partially deficient (Fig. 5a and Supplementary Fig. 17c). In tpl, tpr1, tpr2, tpr3 and tpr4 mutants, AM initiation was not affected in early rosette leaves, but AMs were clearly reduced in tpl tpr1 tpr4 triple mutant, suggesting their overlapping roles in controlling AM initiation (Fig. 5b-d and Supplementary Fig. 17a-b). In contrast to the absence of AMs in loss of function mutants, plants that overexpressed TPL normally formed an AM in per leaf axil (Fig. 6a-b and Supplemental Fig. 16a-c). To monitor TPL expression in early leaf axils, we constructed pTPL::GFP marker lines and found that TPL had a strong signal in the boundary region of emerging leaf primordia, AM and SAM (Fig. 5f and Supplemental Fig. 16a-c). The temporal and spatial patterns of TPL expression confirmed the possibility of TPL regulating AM initiation. Moreover, tpl tpr1 tpr4 triple mutant caused significant reduction of REV and CUC3 expression compared with the WT (Supplemental Fig. 17d).

To characterize the genetic interaction of LCR with TPL, we crossed the p35S::LCR4m to the tpl-1 mutant and obtained LCR4m-OX tpl-1 plants. Phenotype of LCR4m-OX tpl-1 plants was the same as tpl-1, the seedlings failed to form shoot apical meristem and showed various degrees of cotyledon fusion^24,56 (Fig. 6a, c and Supplemental Fig. 17c). Next, we generated the LCR4m-OX TPL plants overexpressed LCR4m and TPL and discovered that overexpression of TPL rescued the AM deficiency of LCR4m-OX, as reflected by REV and STM normal expression in LCR4m-OX TPL (Fig. 6a-c and Supplemental Fig. 16a-c). These observations demonstrate that TPL acted downstream of LCR and as a target of LCR to positively regulate AM initiation via the REV-STM pathway.

miR394 works as a new modulator of AM initiation. miRNAs play a vital role in plant development with a precise control of cell proliferation and differentiation^28,30,34. However, almost no miRNAs role were characterized in AMs until now due to its subtle structure. AMs contain reactivated stem cells and various cell types which make it a representative for tissue development^19,31,43,46. We resorted to tissue specific miRNAs library and identified key regulators of AM initiation (Fig. 1a). Cells expressing miR394s are limited in plants because previous libraries of small RNAs using whole seedlings rarely detected miR394 expression. miR394s expressed with high level in boundary enrichment tissues indicating a role of miR394s in axillary meristem besides SAM. There are two members of miR394s in Arabidopsis, of which miR394b played a dominant role in SAM. miR394b was transcribed in L1 layer of SAM and moved to underneath layers to execute a non-cell-autonomous role in stem cell maintenance. In AMs, miR394a and miR394b collaborate and play overlapped roles in promoting AM initiation, although miR394b mutant displayed a more severe phenotype of AM deficiency (Fig. 1b-e). miR394A expressed strongly in L3 and underneath cells while miR394B showed more signal in L1-L3 (Fig. 2a-b). In situ hybridization indicated that mature miR394s were distributed in gradient in AMs with high expression level in L1 and lower levels underneath (Fig. 2c). In contrast, the LCR showed an opposite expression pattern from L1 to L3 and underneath cells (Fig. 2d-e). Although the transcription of miR394s spread throughout AMs, the gradient of mature miR394s and their targets are similar to that in the SAM. STM and REV expression indicated that miR394s persisted in leaf boundaries to maintain stem cells in younger leaf axils and are increased to activate AM initiation in old leaf axils (Fig. 3a-d). All the results demonstrate that miR394s play a conserved role in stem cell maintenance in both AMs and SAM and promote meristem initiation in axils.

miR394-LCR-TPL mediate a novel pathway regulating AM initiation in Arabidopsis. miRNAs cleave target mRNAs in post-transcriptional level^33,35,36. LCR is a reported target of miR394 and encodes an F-box protein^39,48. Previous studies showed the role of LCR in SAM and leaf development^43,46,47,49. Genetic and cellular experiment indicated that LCR expressed in AMs and negatively regulates AM initiation (Fig. 1, 2 and Supplemental Fig. 5). Transgenic lines with fragment deleted LCR indicated that F-box domain is necessary for LCR function in AMs (Supplemental Fig. 6 and Supplemental Fig. 11). F-box proteins are well known to tightly linked to protein ubiquitylation and degradation^53,57,58, however, the substrates of LCR protein is less known. Here we used IP-MS to screen LCR downstream substrates and identified TPL (Fig. 4a). TPL was reported to control gene transcription with transcription factors as a general repressor^26,27,59. The role of TPL in specific tissues is not deeply studied. Fortunately, phenotyping of tpl and homologous mutants showed significant AM deficiency as LCR OX lines indicating that this family genes participated in AM initiation (Fig. 5b,e). TPL expressed in leaf axils and AMs verified that TPL directly regulates AM initiation in leaf axils (Fig. 5f). Genetic data confirmed the function of LCR and TPL downstream of miR394s (Fig. 6a-c). Together, these results collectively demonstrate a new pathway constituted by miRNAs and F-box mediated proteins that regulate AM formation. This pathway distinguished from traditional pathway that only includes transcription factors. miR394-LCR-TPL play roles from an early stage in boundary and stem cells and continued to AM formation (Fig. 6d).

AM initiation is dynamic and precisely regulated from different aspects. Previous studies focused on the transcriptional control of AMs, the regulation in protein level is less known^{7,8,10,13,14,15,60}. AM initiation is a complicated process through which plants form architecture to reach the highest yield and response to environmental conditions²². Here we first studied AM regulation on protein level. LCR as miR394 target and an F-box protein interacted 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 RNAs^25,33. The interactions of LCR with other proteins such as SE deserve further studies. In this study, we demonstrate that LCR interacted with TPL and accelerated TPL degradation probably through ubiquitylation. LCR itself was strictly regulated in shoot apex, because LCR OX and marker lines constructed by native proteins could not detect signals of LCR. Only mutated versions with miR394 binding sites deleted or F-box domain deleted could be detected in transgenic lines. However, the relationship between miR394 cleavage and F-box regulation, in another word, post-transcriptional and translational control of LCR was not known. The modification sites of TPL and LCR proteins were also not clear. In brief, our results indicated that AM initiation is precisely regulated in transcriptional levels, post-transcriptional levels and protein levels.

Plant materials and treatments.

Arabidopsis thaliana ecotypes Columbia (Col-0) and Landsberg erecta-0 (Ler) were used as wild-type controls. The mir394b-1, lcr-1, tpl, tpr1, tpr2, tpr3, tpr4, tpl tpr1 tpr2, pTPL::TPL and pREV::REV-GR-HA lines are in the Col-0 background^59,61,62,63; the tpl-1, pREV::REV-Venus, pSTM::STM-Venus and p35S::STM-GR lines are in the Ler background^{23,24,54,56,60}. The mir394a/b double mutant was generated by crossing mir394b-1 to mir394a-cr. The mir394a/b lcr-1 triple mutant was generated by crossing mir394a/b to lcr-1. All primers used for mutant genotyping are listed in Supplemental Table S1.

All plants were grown in growth rooms at 22°C under full spectrum white fluorescent light under long day (16 h light/8 h dark) or short day (8 h light/16 h dark) conditions.

Vector construction and plant transformation.

mir394a-cr, mir394b-cr, lcr-cr, p35S::MIR394A, p35S::MIR394B, p35S::LCR, p35S::LCR4m, p35S::LCR5m, p35S::LCR^△F−box, p35S::LCR4m^△F−box, pLCR::LCR, pLCR::LCR4m, pLCR::LCR^△F−box, pLCR::LCR4m^△F−box, pMIR394A::GFPer, pMIR394B::GFPer, gLCR::eGFP, pREV::LCR5m, pKAN1::LCR5m, p35S::TPL and pTPL::eGFP transgenic lines were generated in this study. To generate the plasmids of the above transgenes, the promoter-MIR394A, promoter-MIR394B, promoter-LCR, promoter-TPL, pri-miR394a and pri-miR394b were amplified from Col-0 genomic DNA. The LCR coding sequence (CDS) were amplified from Col-0 cDNA. The LCR4m and LCR5m point mutations were constructed as described earlier^43,47. The LCR^△F−box and LCR4m^△F−box were amplified by two fragments using corresponding primers to removed region F-box.

To generate 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 Bsa1 enzyme (NEB)⁶⁴.To construct overexpression vector, the fused gene of LCR, LCR4m/5m, LCR^△F−box and LCR4m^△F−box CDS and EGFP was cloned into the express vector pCambia1300 driven by the LCR promoter or 35S promoter. The TPL CDS and mCherry was cloned into the express vector pCambia2300 driven by the 35S promoter. To construct expression vector, the miR394a or miR394b promoter inserted into the pMDC204 vector. The LCR or TPL promoter inserted into the pCambia1300 vector. The binary vectors were selected in Agrobacterium-mediated GV3101 transformation to produce transgenic plants in the Col-0 background. Genomic DNA was extracted from T2 transgenic lines, and the primers flanking the designated target site were used for PCR amplification. The PCR products were sequenced and blasted to identify mutation sites. For selection of transgenic plants, seeds were sterilized and germinated on MS medium containing 25 mg/L hygromycin or 50 mg/L kanamycin. Seeds from each transgenic plant were harvested separately for subsequent observation.

Total RNA extraction, qRT-PCR analyses and northern blot.

Total RNA from vegetative shoot apices were collected by 10 days old seedlings or removing the leaves at 30 days after bolting and extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Thermo Fisher). First-strand cDNA was synthesized with 1 ng total RNA by HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and according to the manufacturer’s instructions to used oligo-dT primers. Quantitative real-time PCR (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 using KAPA SYBR FAST qPCR kit (KAPA Biosystems, Beijing, China). ACTIN2 (AT3G18780) were used for normalize the relative expression. The qRT-PCR (Supplementary Table S1) were used to amplify each gene. The mRNA expression levels were calculated by the 2^−△C(t) methods. Data were repeated with three biological and three technical replicates. Data represent means ± SD of three technical replicates.

Small RNA northern blot was performed as described⁶⁵. Total RNA of 10 µg was separated on 15% urea-PAGE gels and then transferred to a nylon membrane for 1 h at 300 mA. The nylon membrane was hybridized overnight at 50°C with 3 'biotin-marked DNA probes that were complementary to the predicted miRNA or U6 sequences. Autoradiography of the membrane was performed using thermo scientific substrate.

RNA sequencing and Small RNA sequencing

Two batches of Col-0, mir394b-1, mir394a-cr, p35S::MIR394A and pLCR::LCR4m vegetative shoot apices (without leaves) in 30 days old were used for total RNA extraction. RNA-seq and small RNA-seq was performed by BerryGenomics (Beijing).

Chemical treatments

For dexamethasone (Dex) treatment²⁰, a 10 µM Dex (Sigma-Aldrich, a 10mM stock of dex solution dissolved in DMSO was diluted in H₂O to give the work concentration.) solution containing 0.01% (v/v) silwet-77 was applied to the leaf axil-enriched shoot apex tissues (under short day conditions growth for 15 days) every two days, after 15 days of continuous treatment, the plants was transferred to long day growth for 30 days, and observation of AM initiation phenotype. For MG132 treatment, The seedlings of 7 days were incubated in a 50 µM MG132 solution (Sigma) or without MG132(0.1% DMSO) for 5h exposed on light. Following the light treatment, the samples underwent dark treatment for 15h and 24h before collected.

Live imaging

For live imaging was performed as previously described protocol^10,66. Briefly, seedlings were grown in MS medium under short day conditions for 15 days and transferred to the dissecting medium (3% agarose) and fixed into a hole using forceps. Leaves between P₅ and P₁₁ were carefully detached from seedlings, laid flat on imaging medium (1/2 MS medium with 1% agarose on the top). FM4-64 (thermo fisher, 10 mg/mL) was applied to the meristem for 20 min to imaging. For time-lapsed live imaging, the leaves were transferred back to new 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 imaging. All live imaging experiments were performed using the water dipping lens of the Olympus (FV1000MPE) confocal laser scanning microscope.

Agarose Sectioning

For agarose sectioning was performed as previously described protocol^15,19. In Briefly, seedlings were grown in soil in short day conditions for 30 days. Shoot apices were collected by removing the leaves, then immediately placed in 2.5% paraformaldehyde (PFA; Sigma-Aldrich) at pH 7.0. After vacuum infiltrated for 30 min on ice and stored overnight at 4°C. Fixed tissue samples were washed with 10% sucrose (prepared with 1% PFA at pH 7.0) for 20 min, with 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. Then, the samples were embedded in 6% low-melting-point agarose (Promega) liquid gel at 37°C and placed at 4°C for 30 min to solidify. Sections of 40µm were prepared using a Leica VT1000S vibratome.

RNA in situ hybridization

RNA in situ hybridization were carried out as described previously⁶⁷. Briefly, 30 days shoot apices without leaves were fixed in 4% paraformaldehyde on ice. After fixation, paraffin embedding were performed as described⁶⁸. Using a Leica (RM2255) microtome, the paraffin blocks were sliced 8 µm and dried overnight at 42°C. After incubation at 37°C for 20 min with protease K digestion, samples were hybridized overnight at 55°C with antisense RNA probes labeled with Digoxegenin (Roche) in a hybridization mix (1.25 ml in situ hybridization salts, 5 ml deionized formamide, 2.5 ml 50% dextran sulfate, 250 µl 50× Denhardt’s solution, 125 µl 100 mg/ml tRNA and 875 µl of nuclease-free H₂O). After washing with 0.2XSSC, probes were detected by incubation with anti-Digoxegenin antibody(0.5µlAnti-DIG-AP་500µl BSA/Triton/TBS for 4 slides) for 2h at RT. Rinse the slide in wash buffer(0.1M Tris-HCl pH9.5, 0.1M NaCl, 50mM MgCl₂) and observe the signal.

cDNA fragments of LCR were amplified and cloned into pEASY-Blunt vector, respectively. In vitro transcription was performed with T3 or T7 RNA polymerase (Roche) in which linearized vectors were added as templates. All primers used for preparing probes are listed in Supplemental Table S1.

Confocal Microscopy, Optical Microscopy, and Scanning Electron Microscopy

Confocal microscopy images were taken with a Zeiss (LSM 980) or a Olympus (FV1000MPE). Excitation and detection wavelengths for GFP, Venus, mCherry were as described^10,15,19. To detect the signal of FM4-64 staining, a 514 nm laser line was used for excitation and a 580–620 nm band-pass filter was used for detection. Autofluorescence was excited at 488 nm and detected in the range of 650-700nm. All images were scanned at 1024 × 1024 pixel resolution. Optical photographs were taken with an Olympus (BX53) microscope equipped with a Canon camera. Scanning electron microscopy was performed by using a HitachiS-4800 field-emission variable pressure scanning electron microscope after standard tissue preparation for cellular observation as previously described²¹.

IP-MS analyses

The immunocomplexes were analyzed by mass spectrometry (MS) at the Chinese academy science institute in botany key laboratory of plant molecular physiology. Briefly, 2 g seedling at 10 days of p35S::LCR4m and p35S::LCR transgenic plants were collected and ground in a mortar using liquid nitrogen. Total proteins were lysed in IP buffer (50 mM Tris-HCI(PH 7.5), 1mM EDTA(PH 8.0), 150 mM NaCl, 2mM MgCl₂, 1mM DTT, 0.1% NP-40,1mM PMSF, 2mM NaF, 50mM MG132 and 1x proteinase inhibitor cocktail (Roche) by gentle rotation at 4°C for 30 min. The supernatant was centrifuged and incubated with GFP-trap beads (ChromoTek) at 4°C for 2-3h. The beads were then washed at least three times using washing buffer (50 mM Tris-HCI(PH 7.5), 1mM EDTA(PH 8.0), 150 mM NaCl, 0.1% NP-40, 1mM PMSF, 2mM NaF, 50mM MG132 and 1x proteinase inhibitor cocktail (Roche)).The GFP-tagged fusion protein complex was isolated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS– PAGE). The target gel lanes was cut and sliced and digestion was carried out with trypsin. After staining and trypsin digestion, the polypeptide was extracted were analyzed by Orbitrap Fusion Lumos (Thermo Fisher Scientific). 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.

Total proteins were extracted from the Arabidopsis tissues in the above buffer. Primary antibodies used in this study include anti-GFP (598, MBL), anti-mCherry (918, MBL), anti-Ubi (ST46-03, Huabio), anti-TPL (A21285, abclonal) and anti-Actin (EASYBIO).

Yeast-two-hybrid screening

LCR, TPL, TPR1, TPR2, TPR3 and TPR4 were amplified from Col-0 cDNA and cloned into Gal4 DNA binding domain (BD) and the Gal4 activation domain (AD), respectively. The vector combinations are listed in the Supplemental Table S1. Standard yeast extract–peptone–dextrose plus adenine (YPAD) media were used and lithium acetate protocol is used to transform digestive plasmids⁶⁹. The yeast strain AH109 using heat-shock treatment (42°C, 15 min) was co-transformed from various bait and prey constructs combinations. The blank construct of prey vector pGADT7 (630442, Clontech) or bait vector pGBKT7 (630443, Clontech) was co-transformed with the bait constructs as a negative control. Equal numbers of co-transformed yeast cells were grown on selective medium SD-Trp/-Leu (630417, Clontech) and SD-Ade/-His/-Trp/-Leu (630428, Clontech) at 30°C for 4 days. The relevant yeast cells grown on selective medium SD-Ade-His-Trp-Leu were diluted and dropped into solid selective medium SD-Ade-His-Trp-Leu respectively containing 50 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) was injected for blue development, and cultured for 2 days for imaging.

BiFC assay

For the bimolecular fluorescence complementation (BiFC) assay, the full length coding sequence of TPL sequence were recombined into the pSPYNE, while the full-length coding sequence of LCR and LCR^△F-box sequence were recombined into the pSPYCE, respectively⁷⁰. All primers used for BiFC are listed in Supplemental Table S1. After verifying the sequence, were introduced into Agrobacterium-mediated of GV3101. The positive transformants were cultured in liquid medium in a injection buffer (10 mM MgCl₂, 10 mM MES, 100 mM acetosyringone) and the overnight culture was diluted to OD600 = 0.5, then different combinations were infiltrated into 4-week-old N. benthamiana leaves as indicated to transiently expressed. Agrobacterium containing H2B-mCherry was used as a nuclear marker. After incubation for 36-48h, the signals of YFP were analyzed by confocal microscopy (Zeiss LSM800 and LSM880).

Data availability

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.

Data availability

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.

Acknowledgements

We thank Xuemei Chen of the Peaking University for miR394 genetic materials, helpful discussions and critical reading for the manuscript. This work was supported by the National Key Research and Development Program of China (2023YFF1001300), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDA24010106 and XDA26030201) and the Hundred Talents Program of CAS to C.Z.

Author contributions

C.Z. and L.L. designed the research; L.L., S.G. T.W. and Z.X. performed the experiments; L.L. and B.H. analyzed the data; and C.Z. and L.L. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence and requests for materials should be addressed to Cui Zhang.

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miR394 and LCR cooperate with TPL to regulate AM initiation

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Abstract

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Introduction

Results

LCR associated with TPL in leaf axils and AMs

Discussion

Methods

RNA sequencing and Small RNA sequencing

Chemical treatments

Live imaging

Agarose Sectioning

Confocal Microscopy, Optical Microscopy, and Scanning Electron Microscopy

IP-MS analyses

Yeast-two-hybrid screening

BiFC assay

Data availability

Declarations

References

Additional Declarations

Supplementary Files

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