Due to the critical role of ABA in the regulation of fruit ripening of strawberry, we evaluated whether MTA affects translation efficiency of other genes in the ABA signaling pathway. We found that genes, such as WRKY DNA-binding protein 40 ( WRKY40), exhibited significant changes in translation efficiency when the MTA was silenced or overexpressed (Additional file 1: Figure S13). This could not be reasonably explained by m 6A deposition because the transcripts of these genes are not m 6A-modified according to our m 6A-seq datasets. We speculate that MTA may regulate translation efficiency of numerous transcripts beyond direct m 6A installation. As expected, MTA repression or overexpression also altered the translation efficiency of a number of ripening-related genes without m 6A modification, such as PG1 relevant to firmness and dihydroflavonol 4-reductase ( DFR) associated with anthocyanin biosynthesis (Additional file 1: Figure S13). All the products from Fruit Shoot are not only free from added sugar. None of their products contains artificial colors and flavorings. The simple juicy and water formula of their drinks provides an accurate balance of sweetness and refreshment. Full size image Evaluation of vegetative characters, sexual characters, fresh fruit firmness, and total sugar content The m 6A-seq was performed according to the method described by Dominissini et al. (2013) [ 37]. Briefly, total RNAs were extracted from the woodland strawberry fruit at S6, RS1, and RS3 stages, and the RNAi- MTA and control fruit by the plant RNA extraction kit (Magen, R4165-02), and then 300 μg of intact total RNAs were used for mRNA isolation by the Dynabeads mRNA purification kit (Life Technologies, 61006). The purified mRNAs were randomly fragmented into ~ 100 nucleotide-long fragments by incubation at 94 °C for 5 min in the RNA fragmentation buffer (10 mM Tris-HCl, pH 7.0, and 10 mM ZnCl 2). The reaction was terminated with 50 mM EDTA, and then the fragmented mRNAs were purified by standard phenol-chloroform extraction and ethanol precipitation. For immunoprecipitation (IP), 5 μg of fragmented mRNAs was mixed with 10 μg of anti-m 6A polyclonal antibody (Synaptic Systems, 202003) and incubated at 4 °C for 2 h in 450 μL of IP buffer consisting of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% NP-40 (v/v), and 300 U mL -1 RNase inhibitor (Promega, N2112S). After the addition of 50 μL Dynabeads protein-A (Life Technologies, 10002A), the mixture was incubated at 4 °C for another 2 h. The beads were subsequently washed twice with high-salt buffer containing 50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40 (v/v), and 0.1% SDS (w/v) and twice with IP buffer. The m 6A-containing fragments were eluted from the beads by incubation with 6.7 mM N 6-methyladenosine (TargetMol, T6599) in IP buffer at 4 °C for 2 h, followed by standard phenol-chloroform extraction and ethanol precipitation. Then, 50 ng of m 6A-containing mRNAs or pre-immunoprecipitated mRNAs (the input) were used for library construction by the NEBNext ultra RNA library preparation kit (NEB, E7530). High-throughput sequencing was conducted on the Illumina HiSeq X sequencer with a paired-end read length of 150 bp following the standard protocols. The sequencing was performed with three independent biological replicates, and each RNA sample was prepared from the mix of at least 60 strawberry fruits to avoid individual differences among fruits. The 1608 hypermethylated m 6A peaks were highly enriched (83.04%) in the CDS region, whereas the 865 hypomethylated m 6A peaks were mainly distributed around the stop codon (40.66%) or within the 3′ UTR (58.59%) (Fig. 3e; Additional file 1: Figure S6). This is in accordance with the dynamic m 6A distribution pattern (Fig. 2), showing that the percentage of m 6A peaks locating in the CDS region increased sharply, while that around the stop codon or within the 3′ UTR declined, from S6 to RS1. Interestingly, of the 1608 hypermethylated m 6A peaks, 1424 (88.56%) fell into the newly generated peaks, which represents ripening-specific peaks (Additional file 11: Table S10). For the differential m 6A peaks identified after ripening initiation, both the hypermethylated and hypomethylated m 6A peaks were highly enriched (over 90%) around the stop codon or within the 3′ UTR (Fig. 3e). To explore the biological significance of genes with dynamic m 6A modification, we performed Gene Ontology (GO) analysis on genes containing differential, non-differential, and ripening-specific m 6A peaks. In line with the progression of fruit development and ripening, genes harboring ripening-specific m 6A peaks were mostly annotated to developmental pathways, including response to hormone stimulus, developmental process, histone modification, small molecular biosynthetic process, and protein transport (Additional file 1: Figure S7a). Similar enrichment was observed for genes covering differential m 6A peaks at the initiation stage of ripening (from S6 to RS1) (Additional file 1: Figure S7b). In contrast, genes with non-differential m 6A peaks were enriched in multiple biological processes in addition to developmental pathways (Additional file 1: Figure S7c). These results suggest that dynamic changes in m 6A modification are responsible for those genes to exert their functions during fruit development and ripening.

The ABA biosynthesis rate-limiting enzyme NCED was reported to play an essential role in the ripening of strawberry fruit [ 34, 38]. Moreover, several critical constituents of ABA signaling, including the ABA receptor FaPYR1 and FaABAR, the type 2C protein phosphatase FaABI1, and the SNF1-related kinase FaSnRK2.6, have been revealed to be indispensable for normal fruit ripening of strawberry [ 57, 58, 59]. Nevertheless, the regulatory mechanisms underlying ABA biosynthesis and signaling pathway remain largely unknown.

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Previous studies reported that the application of low concentrations of cytokinins [ 16, 17], a reduced number of subcultures during the proliferation stage [ 18, 19], and the choice of genotypes [ 20, 21] are critical factors that should be considered when obtaining true-to-type plants. Hence, it is necessary to develop an efficient protocol that can effectively produce genetically stable, virus-free shoots of different cultivars using the meristems. Swartz HJ, Galletta GJ, Zimmerman RH. Field performance and phenotypic stability of tissue culture-propagated strawberries. J Am Soc Hortic Sci. 1981;106:613–67. In Arabidopsis, ABA receptor PYLs could be regulated by post-translational modification, such as phosphorylation [ 60], tyrosine nitration [ 61], and ubiquitination [ 62], which synergistically modulate the abundance and activity of the receptors [ 56]. In contrast, the downstream signal molecules PP2Cs and SnRK2s are mainly controlled by protein phosphorylation and dephosphorylation for maintaining the appropriate ABA signal transduction [ 63, 64, 65, 66]. The transcription of genes in ABA biosynthesis and signaling pathway display dynamic changes in various development processes or in response to environmental stresses [ 56, 67, 68, 69], implying that they undergo precise regulation at transcriptional level. However, little is known about the regulation of genes in the ABA pathway at the post-transcriptional level.

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Notably, hundreds of ripening-induced and ripening-repressed genes, which display significantly higher or lower expression in RS1 compared to S6 (Additional file 12: Table S11), exhibit differential m 6A modification (Additional file 14: Table S13; Additional file 15: Table S14). GO analysis revealed that these genes were enriched in processes such as multicellular organismal development, developmental process, nucleocytoplasmic transport, and anatomical structure development (Additional file 1: Figure S7d), implicating the involvement of m 6A methylation in the regulation of strawberry fruit ripening. Genes in ABA biosynthesis and signaling pathway exhibit differential m 6A methylation upon ripening initiation In all the cultivars, the shoot growth was induced from the meristem cultured on both the medium without Kn (control) and that with low concentration of Kn (0.5 mg L −1), although the induction was stronger in the latter. Surprisingly, no shoot induction was observed in the media containing more than 0.5 mg L −1 (data not shown), which may be due to Kn toxicity to plant cells and tissues. After transferring the explants to the rooting and plant growth medium, the number of shoots that developed per explant was significantly higher in plants cultured on the medium containing Kn 0.5 mg L −1 than in the control. In addition, the shoots induced by 0.5 mg L −1 Kn were likely to be healthier and produced more leaves and roots than those derived from explants grown in the Kn-free medium. This improved growth performance may be explained by the promoting effect of Kn on cell division, leading to higher number of shoots, leaves, and roots. A greater number of surviving shoots and superior plant growth were also observed in explants derived from cultures on the medium with 0.5 mg L −1 Kn during acclimation, which may be attributed to their primarily healthier shoots as compared to those derived from the control. Furthermore, these Kn-derived plants thrived in the greenhouse conditions and were genetically stable in comparison with conventionally propagated plants, as indicated by flow cytometry and RAPD markers. Transient transformation of strawberry fruit mediated by agroinfiltration was performed as previously described [ 54]. To construct the RNA interference (RNAi) vectors, a ~ 300-bp fragment targeting the coding sequence region of MTA or MTB was cloned and inserted into the pCR8 plasmid, and then restructured into the pK7GWIWGD (II) plasmid by using the Gateway LR Clonase TM Enzyme Mix (Invitrogen, 11791-020). To construct the overexpression (OE) vectors, the coding sequence of MTA and MTB was amplified and ligated into the pCambia2300-eGFP plasmid to generate 35S:: MTA-eGFP and 35S:: MTB-eGFP vector, respectively. The resulting constructs were separately transformed into the A. tumefaciens strain GV3101. The agrobacteria were cultured at 28 °C overnight in LB liquid medium supplemented with 50 μg mL −1 kanamycin, 50 μg mL −1 gentamycin, and 50 μg mL −1 rifampicin, and then diluted 1:100 in 100 mL of fresh LB medium to continue culturing for approximately 8 h. The agrobacteria cells were subsequently collected by centrifugation at 5,000 g for 5 min and resuspended in the infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl 2, and 100 μM acetosyringone) to a final OD 600 of 0.8. After being kept at room temperature for 2 h without shaking, the suspensions were injected into the octoploid strawberry fruit at large green (LG) stage by using a 1 mL syringe. The infiltrated fruits were cultured for 5–7 days in a growth room with the following conditions: 23 °C, 80 % relative humidity, and a 16/8-h light/dark photoperiod with a light intensity of 100 μmol m −2 s −1. The experiment was performed with more than three independent biological replicates, and each group contained at least fifteen fruits. The primers used for vector constructions are listed in Additional file 18: Table S17. mRNA stability assay Here we show that m 6A methylation displays a dramatic change at ripening onset of strawberry, a classical non-climacteric fruit. The m 6A modification in coding sequence (CDS) regions appears to be ripening-specific and tends to stabilize the mRNAs, whereas m 6A around the stop codons and within the 3′ untranslated regions is generally negatively correlated with the abundance of associated mRNAs. We identified thousands of transcripts with m 6A hypermethylation in the CDS regions, including those of NCED5, ABAR, and AREB1 in the abscisic acid (ABA) biosynthesis and signaling pathway. We demonstrate that the methyltransferases MTA and MTB are indispensable for normal ripening of strawberry fruit, and MTA-mediated m 6A modification promotes mRNA stability of NCED5 and AREB1, while facilitating translation of ABAR. Conclusion It also combines the essentials of multivitamins. Their Juiced range of Fruit Shoot is the combination of water and juice in the same proportion. They are pressed from juicy, sweet, ripe fruit from the seasonal crops.

If all you are getting from your strawberry plants is runners and no strawberries, see this post to understand the top 10 reasons why that may be happening: Strawberry Plants Producing Runners but no Strawberries?) Compared to the understanding of the molecular basis underlying fruit ripening in climacteric fruits such as tomato, our knowledge regarding the regulation of ripening in non-climacteric fruits, e.g., strawberry, is still limited. Recently, it was elucidated that the ripening of strawberry involves the remodeling of DNA methylation [ 55]. However, it is unclear whether m 6A methylation, which has been revealed to modulate the ripening of tomato fruit [ 36], is involved in the regulation of strawberry fruit ripening. In the present study, we found that m 6A methylation represents a widespread mRNA modification in strawberry fruit and exhibits a dramatic change at the initiation stage of ripening. Overexpression of MTA or MAB, the m 6A methyltransferase genes, promotes fruit ripening, whereas repression of either gene delays ripening (Fig. 6a, b), demonstrating that m 6A methylation participates in the regulation of strawberry fruit ripening. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, et al. N 6-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015;161(6):1388–99. https://doi.org/10.1016/j.cell.2015.05.014.

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Adventitious roots manifest away from the primary roots of a plant, originating instead from the stem, branches, leaves, or old and woody roots. As the name implies, this gives certain plants somewhat of an advantage over other plants. In the case of strawberry plants, they are able to propagate themselves laterally in different directions via runners to find more suitable growing locations for their clone offspring. This allows them to find better soil or areas of better sunlight.

Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m 6A methyltransferase METTL3 promotes translation in human cancer cells. Mol Cell. 2016;62(3):335–45. https://doi.org/10.1016/j.molcel.2016.03.021. Strawberry plants produce runners. These stolons are horizontal stems that run above the ground and produce new clone plants at nodes spaced at varying intervals. Since strawberry plants possess stolons, they are considered “stoloniferous.” The long, leafless stems between the mother plant, plant-growing nodes, and growing tip of the stolon are called “internodes.” Adventitious Roots on a Strawberry Runner In this study, we found that NCED5, ABAR, and AREB1, the genes in ABA biosynthesis and signaling pathway undergo m 6A-mediated post-transcriptional regulation (Fig. 4). The m 6A modifications promote the mRNA stability of NCED5 and AREB1, while enhancing the translation efficiency of ABAR. These findings identify a novel layer of gene regulation in ABA biosynthesis and signaling pathway and establish a link between m 6A-mediated ABA pathway and strawberry fruit ripening. Given the essential roles of ABA in plant development and stress resistance, it is interesting and necessary to explore the regulation of m 6A methylation on these physiological processes. m 6A modification exhibits diverse effects on mRNAs in strawberry Strawberry undergoes an overall loss of DNA methylation during ripening [ 55]. There are four SlDML2 homologs, FveDME1, FveROS1.1, FveROS1.2, and FveROS1.3, in the strawberry genome (Additional file 1: Figure S15a). Our m 6A-seq analysis revealed that FveDME1 and FveROS1.1 contain differential m 6A peaks upon ripening initiation (from S6 to RS1), while FveROS1.2 and FveROS1.3 are not m 6A-modified (Additional file 1: Figure S15b). However, none of these genes showed significantly increased expression from S6 to RS1 (Additional file 1: Figure S15c), suggesting that the homologs of SlDML2 might be dispensable for m 6A-mediated regulation of ripening in strawberry fruit. Previous study has shown that the reprogramming of DNA methylation during the ripening of strawberry fruit is governed by components in the RNA-directed DNA methylation (RdDM) pathway rather than those in the demethylation pathway [ 55]. Our m 6A-seq data indicated that there was no differential m 6A modification in the transcripts of DNA methyltransferase genes in the RdDM pathway during strawberry fruit ripening (Additional file 1: Figure S16). Together, these data suggest that distinct mechanisms underlie the m 6A-mediated ripening regulation in strawberry fruit. m 6A methylation regulates strawberry fruit ripening by targeting ABA pathwayI am very tempted to try propagating from the Snow White runners. I know it’s a risk, but my garden is fairly large and I have a couple of underutilised cold-frames, so I should be able to keep any daughters relatively isolated (from any new plants).