14 publications mentioning mdm-MIR156ad

Open access articles that are associated with the species Malus domestica and mention the gene name MIR156ad. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

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Similarly, the expression levels of some miR156 target genes, including NbSPL2a, NbSPL2b, NbSPL5a, NbSPL5b, NbSPL15a, and NbSPL15b were down-regulated at least 2-fold in 35S:MdMIR156a6 stems, but at least 5-fold up-regulated in 35S:MIM156 stems (Figure 3C).

MxSPL26 is the key target gene in miR156 regulation of adventitious rooting in M. xiaojinensisMiR156 and its targeted SPL genes were demonstrated to control plant phase transition and related traits such as cell size and number, trichome development, anthocyanin synthesis, leafy morphology, flowering time, and lateral root development (Wang et al., 2008; Shikata et al., 2009; Yu et al., 2010, 2015; Gou et al., 2011).

Although, the expressions of PIN and ARF genes were independently from the miR156 expression level, the response of MxPIN1 and MxPIN10 gene expression to auxin treatment differed in Mx-R and Mx-A samples.

Although, miR156 -targeted SPLs are known to control varied physiological and developmental processes (Wang et al., 2008; Shikata et al., 2009; Yu et al., 2010, 2015; Gou et al., 2011), with which the miR156 -targeted SPL gene member is associated with adventitious rooting and how miR156 interacts with other rooting regulatory factors such as IAA are so far not fully understood to date.

Mature miR156 expression levels were significantly up-regulated in 35S:MdMIR156a6 plants and significantly reduced in 35S:MIM156 plants (Figure 3B).

To validate if miR156 regulates adventitious root formation, we generated transgenic tobacco plants expressing 35S:MdMIR156a6 or 35S:MIM156 to enhance or inhibit miR156 activity, respectively (Figure 3A).

Response in MxSPL gene expression to miR156 levelsOf the 13 putative miR156-regulated SPL gene family members in apple genome, nine were actively expressed in both Mx-J and Mx-A plant cuttings, but did not include MxSPL3, MxSPL10, MxSPL11, and MxSPL12.

However, it is not clear which SPL gene involved in regulating adventitious rooting in Arabidopsis, because the expression level of miR156 targeted SPLs had no significantly difference among s pl2/9/11/13/15 mutants, 35S:MIR156A plants and wild-type young seedlings (Xu et al., 2016).

Artificial target mimics were generated by modifying the sequence of the AtIPS1 gene to knock-down miR156 expression (Franco-Zorrilla et al., 2007).

In our previous data, the micro-shoots from adult phase M. xiaojinnesis explants were rejuvenated successfully after 15 passages of in vitro subculture, marked by the elevated expression of miR156 expression in leaves of the micro-shoots, and coupled with recovered adventitious rooting ability and leaf lobes (Xiao et al., 2014).

Therefore, we speculate that decline of miR156 expression in adult phase leafy cuttings may inhibit transcript abundance of RTCS-like gene induced by auxin, thereby reducing the adventitious rooting capacity (Figure 12).

These results are supported by the transcriptome data, which show that PIN and ARF genes in Medicago sativa are not significantly differentially expressed between miR156 overexpression and wild-type plants (Gao et al., 2016).

In comparison to the wild-type samples, NbPINs and NbARFs expressions was not substantially modulated in 35S:MdMIR156a6 and 35S:MIM156 plants (Figures 7, 8), indicating that miR156 promoted adventitious root formation independently from changing PIN and ARF genes expression.

These data suggest that high expression level of miR156 is required for auxin inducing expression of RTCS-like during adventitious root formation in M. xiaojinesis and transgenic tobacco.

To check whether miR156 affects the initiation of adventitious root primordia, serial cross sections of the stems of wild-type, 35S:MdMIR156a6 expressing transgenic lines, and 35S:MIM156 expressing transgenic tobacco plants were stained with toluidine blue.

Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3.

ARF genes expression did not change with miR156 levelsThe MxARF4, MxARF7, MxARF14, MxARF15, MxARF16, MxARF17, MxARF19, MxARF20, and MxARF28 genes showed higher expression in Mx-A than in Mx-R cuttings with or without IBA treatment during the adventitious root formation process (Supplementary Figure 6).

However, the relationship between miR156 and PIN gene expression was not robust in transgenic tobacco; no distinct changes were detected in the expression of any NbPIN members between wild-type, 35S:MdMIR156a6, and 35S:MIM156 tobacco plants (Figure 7C).

PIN genes are not altered by miR156 expressionThe expression levels of MxPIN3, MxPIN4, MxPIN6, MxPIN8, MxPIN9, MxPIN12, and MxPIN13 did not show obvious differences between Mx-A and Mx-R leafy cuttings during the adventitious root-induction process (Figure 7A and Supplementary Figure 4).

Similarly, miR156 expression was not affected by IBA application during the induction of adventitious root development in both juvenile and adult E. grandis stem cuttings (Levy et al., 2014).

RTCS-like gene expression varied with miR156 levelsIn M. xiaojinensis, the MxRTCS-like gene was up-regulated 6–24 h after IBA treatment in both Mx-A and Mx-R cuttings, but the maximum induction of MxRTCS was observed in Mx-R cuttings at 120–168 h, which was 4-fold higher than that measured in Mx-A cuttings (Figure 9).

Of the 13 putative miR156-regulated SPL gene family members in apple genome, nine were actively expressed in both Mx-J and Mx-A plant cuttings, but did not include MxSPL3, MxSPL10, MxSPL11, and MxSPL12.

Hence, auxin inducing MIR156 and SPL expression may be tissue/organ specific during lateral root development.

Profiling microRNAs in Eucalyptus grandis reveals no mutual relationship between alterations in miR156 and miR172 expression and adventitious root induction during development.

MxSPL26 is the key target gene in miR156 regulation of adventitious rooting in M. xiaojinensis.

MiR156 and its targeted SPL genes were demonstrated to control plant phase transition and related traits such as cell size and number, trichome development, anthocyanin synthesis, leafy morphology, flowering time, and lateral root development (Wang et al., 2008; Shikata et al., 2009; Yu et al., 2010, 2015; Gou et al., 2011).

miR156 affects adventitious root formation independently from PIN and ARF genes expressionAuxin and miR156 are both involved in adventitious root development (Zhang et al., 2011; Feng et al., 2016; Steffens and Rasmussen, 2016; Massoumi et al., 2017).

Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156 -targeted SPL transcription factor.

Conversely, auxin can induce the expressions of two MIR156 genes and two SPL genes during lateral root development in transgenic Arabidopsis (Yu et al., 2015).

This indicates that miR156 may not be associated with the expression of ARF transcription factors during the regulation of adventitious root formation.

Figure 2Expression level of miR156 in adult phase (Mx-A), juvenile phase (Mx-J), and rejuvenated (Mx-R) leafy cuttings of Malus xiaojinensis.

ARF genes expression did not change with miR156 levels.

Auxin and high miR156 expression level are both necessary for adventitious rooting.

Similarly, tomato and tobacco plants overexpressing miR156 exhibit dense aerial roots on their stems, while none appear on the stems of wild type plants (Zhang et al., 2011; Feng et al., 2016).

Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

The relationship between miR156 expression and adventitious root formation.

In adult leafy cuttings, decline of miR156 caused high expression level of SPL26.

In the Congrass1 maize mutant that overexpresses miR156, prop roots are produced at all nodes in the plant, while these roots only grow from shoot-born meristem at the juvenile nodes in wild-type plants (Chuck et al., 2007).

Supplementary Figure 8Diagram of the miR156 target sites of the WT and modified version of MxSPLs.

Response in MxSPL gene expression to miR156 levels.

Developmental functions of miR156-Regulated Squamosa promoter binding protein-like (SPL) Genes in Arabidopsis thaliana.

To evaluate how juvenility mediates adventitious rooting in woody plants and using apple rootstock M. xiaojinensis as an example, we analyzed the transcript level of miR156, miR156 putative target SPL gene expression, and MxPIN and MxARF gene family members during the adventitious rooting process.

RTCS-like gene expression varied with miR156 levels.

In the present study, the miR156 expression level was manipulated in transgenic tobacco to analyze how miR156/SPL modules are involved in adventitious rooting.

Dual effects of miR156 -targeted SPL genes and CYP78A5/KLUH on plastochron length and organ size in Arabidopsis thaliana.

Supplementary Figure 12Phylogenetic analysis of miR156 -targeted SPL between apple and Arabidopsis.

Elevated levels of miR156 promote adventitious root formation in maize, tomato, and tobacco, indicating that SPL proteins inhibit adventitious root formation (Chuck et al., 2007; Zhang et al., 2011; Feng et al., 2016).

In apple seedlings, the glutathione content and glutathione/glutathione disulfide ratio were much higher in the juvenile phase than in the adult phase, and modulating glutathione content caused concomitant changes in miR156 expression levels (Du et al., 2015).

Consistent with the rooting ability, the expression level of miR156 in Mx-J and Mx-R cuttings were significantly higher than that in Mx-A cuttings (Figure 2).

The expression level of miR156 was decreased during juvenile to adult phase change (Du et al., 2015; Ji et al., 2016).

Twenty-seven SPL gene family members were identified in the apple genome and 13 were predicted to be targets of miR156 using degradome sequencing (Li et al., 2013; Xing et al., 2014).

Thus, miR156/SPL modules were not downstream targets of auxin during adventitious root formation.

PIN genes are not altered by miR156 expression.

The infected leaves were selected on MS medium supplemented with 100 mg/L kanamycin and 300 mg/L cefotaxime sodium to generate rSPL and miR156 co -overexpressing transgenic lines.

In conclusion, in semi-lignified leafy cuttings of M. xiaojinensis, a relatively higher expression of the miR156 is necessary for adventitious root formation.

In the present study, we found that the high miR156 expression was required for adventitious roots formation in an apple rootstock, M. xiaojinensis.

The juvenile to adult phase change is initiated by a decrease in the expression of the miR156.

However, the uncoupling of miR156 expression and adventitious root formation was reported in E. grandis (Levy et al., 2014); therefore, miR156 may be necessary but not sufficient for adventitious rooting in woody plants.

2017.01059/full#supplementary-material Supplementary Figure 1Expression profiles of nine miR156 precursors were analyzed using semi-quantitative RT-PCR in Malus xiaojinensis stem bark.

These results provided a potential strategy for the improvement of the adventitious rooting ability of perennial woody plants via manipulating miR156 and SPL gene expression.

miR156 affects adventitious root formation independently from PIN and ARF genes expression.

However, the molecular mechanism underlying miR156 modulate auxin induced RTCS-like gene expression remains unknown.

Correlation analysis of miR156 expression and redox status during the phase change of Malus xiaojinensis seedlings.

Indeed, miR156 expression levels were significantly higher in Mx-J and Mx-R cuttings than Mx-A cuttings (Figure 2), and the rooting ability of Mx-R and Mx-J cuttings were consistently higher than that of Mx-A cuttings (Figure 1).

Except for high expression of miR156, auxin was also integrant for adventitious root formation.

These results suggest that miR156 and miR156 putative targeted MxSPL genes may play important roles in adventitious root formation during the juvenile to adult phase change.

A significant decrease in miR156 expression was observed in shoots taken from 1.4 m trunk above ground compared to shoots taken from below 1.4 m in M. xiaojinesis seedlings (Ji et al., 2016).

Similarly, the auxin response was not changed in either Pro35S:MIR156 or Pro35S:MIM156 Arabidopsis, indicating that miR156 does not modulate the auxin response during regulating shoot regeneration (Zhang T. Q. et al., 2015).

MiR156 expression level was analyzed by qRT-PCR as described previously (Xiao et al., 2014).

MiR156 acts by repressing the expression of a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes.

Auxin and miR156 are both involved in adventitious root development (Zhang et al., 2011; Feng et al., 2016; Steffens and Rasmussen, 2016; Massoumi et al., 2017).

However, the interaction of miR156 and auxin signaling pathway is unexplored during adventitious root development.

The miR156-resistant SPLs (rSPLs) were made by two rounds of mutagenic PCR using KOD-Plus Nero DNA polymerase (KOD-401, TOYOBO LIFE SCIENCE, Japan), and sequencing confirmed the mutations by BGI (Shenzhen, China).

Temporal control of trichome distribution by microRNA156 -targeted SPL genes in Arabidopsis thaliana.

MiR156 functions via its target gene MxSPL26 and rooting-related genes such as MxRTCS-like; but acts independently of MxPIN or MxARF family members in response to auxin.

The role of miR156/SPLs modules in Arabidopsis lateral root development.

To determine whether miR156 regulates adventitious root formation through modulating endogenous auxin levels or auxin polar transport, we examined adventitious rooting capacity in wild-type, 35S:MdMIR156a6, and 35S:MIM156 stem cuttings grown on MS medium supplemented with different concentrations of indole-3-acetic acid (IAA) or 1-N-naphthylphthalamic acid (NPA).

The results elucidate the role of miR156 in adventitious root formation and will be useful in horticultural and forestry industries.

Collectively, these findings suggest that miR156 does not modulate the early auxin response.

Azacytidine and miR156 promote rooting in adult but not in juvenile Arabidopsis tissues.

When miR156 level was manipulated via transformation with a 35S:MIR156 construct in tomato, tobacco, or Arabidopsis, the adventitious rooting increased (Zhang et al., 2011; Feng et al., 2016; Massoumi et al., 2017).

However, during the adventitious root formation in recalcitrant woody plants, whether and how miR156 interacts with auxin remains unknown.

Then, the function of miR156 in adventitious root formation was validated by generating transgenic tobacco lines.

To investigate which M. xiaojinensis SPL gene family member was involved in adventitious root formation, mutants of SPL genes, indicated here as resistant SPLs (rSPLs), were designed to no longer be targeted by miR156 (Supplementary Figure 8; Schwab et al., 2005).

Interaction between miR156 and auxin during adventitious root formation.

In Arabidopsis, maize (Zea mays) and various woody plants including Acacia confusa, Acacia colei, E. globulus, H. helix, Quercus acutissima, and Populus × Canadensis, miR156 is highly abundant in seedlings and decreases in adult plants (Wu and Poethig, 2006; Chuck et al., 2007; Wang et al., 2011).

Modulation of miR156 to identify traits associated with vegetative phase change in tobacco (Nicotiana tabacum).

Nine miR156 precursor genes were previously identified in apple genome (Ma et al., 2014).

However, the effect of miR156 on adventitious root formation barely rated a mention in previous reports (Zhang et al., 2011; Feng et al., 2016; Massoumi et al., 2017).

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To test whether the changes in miR156 expression level may affect redox homeostasis, an expression construct containing the miR156 precursor MdMIR156a6 under the control of the Cauliflower mosaic virus 35 S promoter, and a target mimic, consisting of a non-cleavable RNA that formed a non-productive interaction with a complementary miR156 (MIM156) to inhibit the activity of miR156, were genetically transformed into Nicotiana benthamiana.

The microRNA miR156 regulates the vegetative phase change via inhibition of the target SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes 8, 9. In Malus species, during phase change, miR156 expression declines at about the 80th node and attains a minimum level at about the 140th node; on the other hand, during rejuvenation, miR156 levels are recovered in rejuvenated in vitro shoots after several subculture cycles 6, 10.

MdMIR156a6 overexpression transgenic plants accumulated much higher quantities of mature miR156 than the WT plants, whereas miR156 expression was greatly inhibited in the MIM156 transgenic plants (Fig.   6B).

The reduced levels of miR156 during phase change were also concomitant with significantly and robustly increased expressions of its targets, MdSPL26, MdSPL23, and MdSPL10&11 (Fig.   S1).

In Atrbohd mutant, the concentration of H [2]O [2], GSH, expressions of miR156 and AtSPLs did not differ significantly from the wild-type, in Atrbohf mutant, however, there was a significant decrease of GSH content, GSH/GSSG ratio and miR156 transcripts, the expression of AtSPL9 correspondingly increased (Figs  S9, S10).

These results prompted us to examine the expressions of miR156 targets MdSPL26, MdSPL23, and MdSPL10&11, which responded to phase change.

The relative expression level of miR156 in the MED, DPI, and BSO treatments were significantly decreased on days 2–6, whereas a dramatic increase in miR156 expression level was observed in the OTC treatment except on day 6 (Fig.   4C).

The present results showed that of the seven family members predominantly expressed in leaf and shoot tip tissues, only transcript abundance of MdMIR156a5 and MdMIR156a12 were significantly higher in juvenile than adult phases, which is consistent with the dynamics of miR156 expression.

To validate the changes in miR156 in response to GSH or redox homeostasis alteration, an ideal experimental system with constant and robust miR156 expression is necessary, because in plant the ROS or GSH levels and miR156 expression varied with the process of vegetative phase change and as well subculture cycles during rejuvenation 6, 10.

To get better convincing data, nine hybrid trees were used for analyzing the variations in MdHXKs expression patterns between juvenile and adult phase and to consider if MdHXKs were co-expressed with miR156.

In Arabidopsis thaliana, for example, auxin induces expression of MIR156B and MIR156D in roots and subsequently increases miR156 expression during lateral-root growth [57].

Consequently, the changes in miR156 levels were concomitant with the expression of the dominant ontogenesis responsive miR156 target, MdSPL26, which is highly consistent with our previous findings in M. xiaojinensis [17].

Relative expression levels of miR156, MdHYL1, MdMIR156 and MdSPL family membersThe expression levels of mature miR156 were analyzed using the method described by Du et al. [10].

Overexpression of miR156 in transgenic Populus × canadensis reduces the transcripts of miR156 -targeted SPL genes and drastically prolongs the juvenile phase [16].

Though the expressions of both pri-MIR156A and mature miR156 are much higher during growth in mir159ab mutant than in wild type Col-0, but they still decline with days after planting, which indicates an independent mechanism is more powerful on regulating vegetative phase change [61].

It seems that the H [2]O [2] levels are not a direct factor than GSH affecting miR156 expression, but the exact relationship between ROS, GSH and miR156 remains unclear.

These data indicate that miR156 expression is, at least partially, under transcriptional regulation, and that MdMIR156a5 and MdMIR156a12 are key family members responsive to ontogenetic cues.

The present results are strongly supported by an analogous observation that the developmental stage associated decline in miR156 expression level is partially mediated by sugar at the transcriptional level of MIR156A and MIR156C in Arabidopsis [35].

To understand whether the phase change -associated decrease in miR156 expression level is due to transcriptional regulation by redox homeostasis in plants, in this study on apple (Malus domestica) we first confirmed the ontogenesis-related members of the MdMIR156 gene family.

However, the Arabidopsis mutant gin2-1, which lacks HEXOKINASE 1 (HXK1), is only slightly precocious in the transition to the adult phase, and thus sugar may not be the only factor that regulates miR156 expression [35].

Then, we have previously validated that MdMIR156a6 is one of the coding genes for miR156 precursor, because several NbSPLs were down-regulated in 35 S: MdMIR156a6 transgenic tobacco lines, but increased in 35 S:MIM156 lines [17].

Conversely, whereas miR156 transcript level was modulated by transformation with MIM156 and MdMIR156 overexpression, the H [2]O [2] and GSH concentration in transgenic N. benthamiana plants did not change consistently, which suggested that H [2]O [2] and GSH are involved upstream of the miR156 transcriptional regulatory network.

Thus the impacts of GSH on miR156 expression were established.

Defoliation disrupts miR156 expression, which implies that a mobile signal(s) is (are) derived from the pre-existing leaves 29, 30.

In ‘ Orin’ suspension cells, the redox parameters and the expression of miR156 remained constant and robust with successive subculture cycles (Figs  S2– S4).

In the preliminary experiments, in suspension cells derived from apple ‘ Orin’ leaves, mature miR156 expression level and redox parameters remained constant and robust with successive subculture cycles.

In DPI treated in vitro shoots, a slight but statistically significant increase in GSH was detected, a drastic increase in miR156 expression was observed.

The expression levels of mature miR156 were analyzed using the method described by Du et al. [10].

To verify that MdMIR156 genes can be processed into mature miR156, the expression constructs MdMIR156 (a5 or a12) and MdSPL (MdSPL7 - MDP0000170630 or MdSPL26- MDP0000142582) were mixed and transformed simultaneously into Nicotiana benthamiana by injection [79].

The data showed obviously that the miR156 expressions were significantly higher in the juvenile phase, which was consistent among all the nine hybrids (Fig.   S8).

The transcripts of MdMIR156a5 and MdMIR156a12 exhibited significantly lower levels in the adult phase than in the juvenile phase, which was closely correlated with mature miR156 expression (Fig.   1B).

In apple seedlings, the expression of miR156 decreased gradually during ontogenesis.

When the H [2]O [2] or GSH concentrations of suspension cells were altered by BSO or OTC treatment, the levels of MdMIR156a5 and MdMIRa12 transcripts as well as the mature miR156 expression level changed correspondingly.

In the apple genome, of the 31 putative genes that encode precursors of miR156, MdMIR156a5 and MdMIR156a12 responded to ontogenetic regulation, and these two genes were transcriptionally regulated downstream of redox signals such as GSH.

In the present study, we used suspension cells to avoid variation in miR156 expression with successive subculture cycles.

We thus also suspected that the variations in MdHXKs expressions between ontogenetic phases were caused by the factors independent with miR156 levels, which agreed to the postulation by Yang and the colleagues [35].

In the present experiment, the H [2]O [2] concentration of suspension cells was either enhanced in response to MED or inhibited by DPI treatment, whereas both GSH concentration and miR156 transcript level declined significantly.

Sucrose inhibits pri-miR156 transcription and processing.

Subsequently we measured the concentration of H [2]O [2], GSH miR156 and miR156 -targeted AtSPL genes (AtSPL3 and AtSPL9) expression in Atrbohd and Atrbohf mutant.

In this study, changes in GSH level and miR156 expression were closely correlated, but we did not find significant and consistent variations in sugar contents and TPS enzyme activity between the juvenile and the adult phase in three apple hybrid trees.

The H [2]O [2] and GSH concentrations showed no regular differences between the MdMIR156a6 -overexpressing and miR156 -mimetic transgenic lines, and also between the WT and transgenic lines (Fig.   6C).

Twenty-seven gene family members containing SPL-box were identified in the apple genome, 14 of which were putative targets of miR156 (MdSPL3, 4, 6, 7, 10, 11, 12, 18, 20, 21, 22, 23, 24, 26).

These data from Arabidopsis further support that GSH, but not H [2]O [2], affects miR156 expression.

Three independent MdMIR156a6 overexpression transgenic lines (p35S: MdMIR156-1, p35S: MdMIR156-2, and p35S: MdMIR156-3) and three miR156 mimetic transgenic lines (MIM156-1, MIM156-2, and MIM156-3) were chosen for further analysis.

Figure 4Changes in hydrogen peroxide (H [2]O [2]) concentration (A), glutathione (GSH) content, glutathione/glutathione disulfide (GSH/GSSG) (B) and relative expression of mature miR156 (C) of suspension cells of apple ‘ Orin’ leaf treated with redox-modulating chemicals.

Relative expression levels of miR156, MdHYL1, MdMIR156 and MdSPL family members.

To date, there are no data illustrating the relationship among sugar, GSH and miR156 expression.

These data indicated that the change in miR156 expression level did not affect H [2]O [2] and glutathione concentrations constantly.

These results indicated that transcripts of MdMIR156a5 and MdMIR156a12 may degrade MdSPLs, the target of miR156, and thus are indicated to be precursor genes of mature miR156.

These changes were consistent with the changes in miR156 expression level.

In both leaf and shoot tip samples, the expression level of mature miR156 in the adult phase was dramatically lower than that in the juvenile phase (Fig.   1A).

Figure 1Quantitative expression of miR156 (A) and major members of the MdMIR156 gene family (B) in shoot tips and leaves in the juvenile (J) and adult (A) phase of three individuals of Malus asiatica ‘Zisai Pearl’ ×  M. domestica ‘Red Fuji’.

When H [2]O [2] concentrations of in vitro shoots of an apple seedling are manipulated with menadione (MED) or diphenyleneiodonium (DPI) treatment, the concentrations of GSH was observed extremely lower in MED treated in vitro shoots than in untreated control, and the relative expression of miR156 decreased to a significantly low level.

Taken together, these results indicated that miR156 was regulated downstream of ROS and GSH.

miR156 is under transcriptional regulation during phase change.

This finding is insufficient to exclude the contribution of post-transcriptional processing of primary miR156 precursors on the decline in mature miR156, but it does not conflict with the hypothesized transcriptional regulation of miR156 during the vegetative phase change.

When GSH contents were altered with L-2-oxothiazolidine-4-carboxylic acid (OTC) or buthionine sulphoximine (BSO) application, miR156 expression varies concomitantly, but no substantial changes in H [2]O [2] concentrations were detected in OTC and BSO treated in vitro shoots compared to the control [10].

miR156 is regulated downstream of GSH and ROS.

Thus, when suspension cells were treated with exogenous redox modulators, the changes in transcript levels of MdMIR156a5, MdMIR156a12, and mature miR156 were consistent with the changes in reduced GSH concentration and the GSH/GSSG ratio, and showed no direct correspondence with H [2]O [2] concentration.

But in all cases, miR156 levels varied with GSH concentration and GSH/GSSG ratio but not directly with H [2]O [2] concentration [10].

In our previous experiments, in which in vitro shoots were treated with redox-modulating chemicals, miR156 transcript levels varied with GSH concentration and GSH/GSSG ratio but not with H [2]O [2] concentration [10].

Therefore, both GSH concentration and thus miR156 transcript level declined significantly (Fig.   S11).

On the other hand, GSH levels were altered by OTC or BSO treatment, MdMIR156s and mature miR156 transcription varied with GSH concentration, but H [2]O [2] concentration did not change throughout the experimental period.

Prior to the current study, only one MdMIR156 precursor (MdMIR156h) has been experimentally verified to be processed into mature miR156 [56].

In the apple genome, there are 31 putative MdMIR156 genes encode pre-miR156 (Fig.   S5, Table  S7).

Recently, in Arabidopsis, miR159 has been found to modulate vegetative phase change upstream of miR156 through MYB33 [61].

We confirmed in this study MdMIR156a5 and MdMIR156a12 can also be transcribed and processed into mature miR156 by using the same approaches.

In Atrbohf mutant, the miR156 transcription also changed with GSH concentration.

Given that not all the members of the MdMIR156 gene family responded to ontogenetic signals, additional functions of miR156 may be unexplored.

The ontogenetic signal upstream of miR156 is proposed to originate from leaf primordia.

In response to OTC or BSO treatment, the transcription of MdMIR156a5, MdMIR156a12 and thus mature miR156 varied depending on GSH concentration and GSH/GSSG ratio.

During the vegetative phase change in apple, ROS accumulate and miR156 transcription declined.

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The expression patterns of these miRNAs and their targets could be divided into four types: (1) mdm-miR156, mdm-miR160, mdm-miR535 and their targets, the SBP, AP2, AP2-like, ARF16, AFB, DC19 and RD19 genes, had the highest expression levels in roots but relatively low expression levels in fruit (Figure  9A,B,D,F and H); (2) mdm-miR393, mdm-miR398a, mdm-miR398b and their targets, the SPL2, SPL9 and ACA8 genes, were found to be expressed most abundantly in flowers but had relatively low expression levels in fruit and roots (Figure  9A,C,E,F,G and H); (3) mdm-miR172, mdm-miR162, mdm-miR162, mdm-miR5225 and their targets, the ARF17, LETM1-LIKE and ADH2 genes, showed high expression levels in leaf tissue but relatively low levels were observed in stems (Figure  9B,D,E,G and I); (4) mdm-miR858 and mdm-miR3627 and their targets, the TIR1 and MYB5 genes, had high expression levels in fruit but low levels in stems (Figure  9C,G and J).

The increased expression of miRNA156 and decreased expression of its targets (SPLs) delayed flowering, whereas inhibiting miR156 expression accelerated flowering [43, 44].

Additionally, mdm-miR156 was highly expressed in roots but had almost no expression in flowers; however, its targets, SPL2 and SPL9, were more highly expressed in flowers than in other tissues (Figure  9A).

Additionally, miR156 regulated its targets, the SPL family, through translational inhibition and gene silencing in A. thaliana[39].

The up-regulation in J compared with A was confirmed for mdm-miR156 during leaf development (Figure  7A), while its targets, SPL2 and SPL9, showed higher expression levels in A than in J from March to August, with their levels gradually increasing (Figure  7A).

mdm-miR156 had significantly higher expression levels in younger (except 1-year-olds) than in older tree leaves (4, 5, and 6 years old) (Figure  8A), whereas its targets (SBP, SPL2 and SPL9) had relatively higher expression levels in older tree leaves (5 and 6 years old) than in younger tree leaves (1 and 2 years old) (Figure  8B).

Our results showed that leaves in the juvenile phase were much smaller and had higher miRNA156 expression levels and lower SBP, SPL2 and SPL9 expression levels compared with adult leaves, implying that miRNA156 and its targets may play important roles in the juvenile to adult transition, leaf development and the transition to flowering.

The overexpression of miR156 in transgenic Populus ×  canadensis reduced the expression of miR156 -targeted SPL genes and miR172, and drastically prolonged the juvenile phase [1].

Some genes involved in flowering were regulated by the expression levels of miRNA156 and their targets in plants.

These data suggest that the expression patterns of miRNAs and their targets, such as miR156 and SPL9, display opposite trends during leaf development.

For example, in Arabidopsis thaliana, high levels of miR156 reduced the expression levels of SPL TFs, which activated SUPPRESSOR of CONSTANS 1 (SOC1), LEAFY (LFY), AGAMOUS-LIKE 42 (AGL42), FRUITFULL (FUL) and APETALA1 (AP1) genes that regulate the transition from juvenile to adult phase [14].

The miRNAs within cluster 1 (miR172 for A and J) typically displayed high expression levels during the later stages of leaf development (August), but miRNAs within cluster 6 (miR156, 169, 393 and 858 for A and miR156 and 5225 for J) displayed opposite results, with high expression levels during the early stages (April and May) (Figure  7 and Additional file 12A).

The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition.

Compared with the expression profile of mdm-miRNA156, mdm-miR172 showed a high expression level in older tree leaves (4-, 5- and 6-years-old) and this expression increased gradually in 1- to 6-year-olds (Figure  8A,B).

Analyzing known miRNA expression levels between the A and J libraries revealed that the 17 known miRNA families (mdm-miR156, 172, 398, 397, 7125, 408, 160, 7124, 393, 3627, 5225, 396, 858, 535, 162, 2118 and 7120) were differentially expressed (Figure  5).

Expression levels of miRNA156 and their targets were associated with the transition from vegetative to reproductive growth and the transition to flowering.

It was reported that miR156 regulated leaf development, showed juvenile characteristics when overexpressed in plants and had reduced expression levels in adult leaves [12, 38].

The expression of miRNAs and their targets in leaves of different ages: mdm-miR156 (A); mdm-miR172 (B); mdm-miR393 (C); mdm-miR160 (D); mdm-miR162 (E); mdm-miR535 (F); mdm-miR3627 and mdm-miR5225 (G); mdm-miR398a (H); mdm-miR398b (I); and mdm-miR858 (J).

miR156 family numbers (A) and other known miRNA family numbers (B) expressed higher in J than in A; miR172 family numbers (C) and other known miRNA family numbers (D) expressed higher in A than in J. Putative novel miRNA in M. hupehensisThe reference genome sequences of the domesticated apple (Malus × domestica Borkh. )

The expression of miRNAs and their targets in A and J leaves: mdm-miR156 (A); mdm-miR172 (B); mdm-miR393 (C); mdm-miR160 (D); mdm-miR162 (E); mdm-miR535 (F); mdm-miR3627and mdm-miR5225 (G); mdm-miR398a (H); mdm-miR398b (I); and mdm-miR858 (J).

Expression of miRNAs and their targets in different tissue: mdm-miR156 (A); mdm-miR172 (B); mdm-miR393 (C); mdm-miR160 (D); mdm-miR162 (E); mdm-miR535 (F); mdm-miR3627 and mdm-miR5225 (G); mdm-miR398a (H); mdm-miR398b (I); and mdm-miR858 (J).

miR156 family numbers (A) and other known miRNA family numbers (B) expressed higher in J than in A; miR172 family numbers (C) and other known miRNA family numbers (D) expressed higher in A than in J. The reference genome sequences of the domesticated apple (Malus × domestica Borkh. )

Previous research also showed that SPL9 was expressed in the vegetative shoot apices, although the expression level of miRNA156 was almost undetectable [42].

For example, miR156 regulates FLOWERING LOCUS T expression in apical meristem to control temperature-responsive flowering in A. thaliana[45].

In this study, we confirmed the up-regulation of mdm-miR156 in J leaves compared with A leaves during leaf development (Figure  7A).

For example, the targets of mdm-miR156 and mdm-miR160 were associated with growth and flower development in plants.

It was reported that GA accelerates flowering through the degradation of transcription repressors, DELLAs, and that DELLAs directly bind to miRNA156 -targeted TFs (SPL family members), which promote flowering by activating miR172 and MADS-box genes [21].

The known miRNA targets included some TFs, including SPL2 (mdm-miR156), SPL9 (mdm-miR156), ARF16 (mdm-miR160) and MYB5 (mdm-miR858), and others contained several regulatory proteins, including the LETM1-LIKE protein (mdm-miR162), AUX signaling F-box 2 protein (mdm-miR393) and the AT hook motif DNA -binding family protein (mdm-miR3627) (Table  3).

mdm-miR156 is highly abundant in J leaves and decreases in A leaves, while mdm-miR172 has the opposite expression pattern in the two leave types.

Others, including mdm-miR156, 166, 167, 168, 408 and 391, had high expression levels, with read counts that reached more than 10,000 in each library (Additional file 4).

In total, 127 targets of 25 known miRNA families, including mdm-miR156, mdm-miR159, mdm-miR166 and mdm-miR172, were detected in our library (Table  3; Additional file 6).

Among these, the expression levels of mdm-miR156 and 11 other miRNA family members, mdm-miR160, 7124, 393, 3627, 5225, 162, 2118, 7120, 396, 858 and 535, in the J library were significantly higher than in the A library (Figure  5A,B).

This was consistent with our results that the expression level of mdm-miR156 in the J library was significantly higher than in the A library (Figure  5).

For example, mdm-miR156 regulated 15 genes, including the SBP domain, SPL2, SPL9 and acyl-CoA synthetase5.

miR156 acts in several pathways that control different aspects of vegetative development and play an important role in the juvenile phase [12].

A majority of the 42 known miRNA families had several members, and five families, mdm-miR156, mdm-miR171, mdm-miR172, mdm-miR167 and mdm-miR399, had 31, 15, 15, 10 and 10 members, respectively.

Our results showed that the juvenile to adult phase transition and flowering were controlled by mdm-miRNA156 and mdm-miRNA172.

4

However, in treatment group of “Starkrimson,” the expression levels of miR156 continuously decreased from 0 h to 6 days and in the “Starkrimson” control group, the expression levels of miR156 were slightly fluctuated at low expression level.

The expression profiles of MdSPL9 (MDP0000297978), MdbHLH (MDP0000225680), MdMYB9 (MDP0000210851), and MdANR2 (MDP0000320264) target genes regulated by mdm-miR156, mdm-miR828, mdm-miR858 and miR5072 were analyzed, respectively.

It has been reported that miR828 and miR858 could directly or indirectly control anthocyanin biosynthesis in apple, and differentially expressed miR156 can positively regulate anthocyanin biosynthesis by the SPL transcription factor in Arabidopsis thaliana (Gou et al., 2011; Xia et al., 2012).

In addition, the expression of target gene MdSPL was a perfect inverse to that of mdm-miR156 after 1–2 days debagging in “Granny Smith.

”Target genes of miR156 have been known as SPL transcription factors in Arabidopsis, which are exclusively expressed in the shoot apex (Cardon et al., 1999; Schwarz et al., 2008).

” Target genes of miR156 have been known as SPL transcription factors in Arabidopsis, which are exclusively expressed in the shoot apex (Cardon et al., 1999; Schwarz et al., 2008).

For example, we found that miR160, miR156, miR171, miR172, miR395, and miR398 were differentially expressed upon debagging, these families are predicted to be regulated by UV-B radiation in Arabidopsis and juvenile maize leaves, respectively (Zhou et al., 2007).

Negative Regulation of anthocyanin biosynthesis in Arabidopsis by a miR156 -targeted SPL transcription factor.

The expression levels of four known miRNAs (mdm-miR156, mdm-miR828, mdm-miR858, and miR5072) and their target genes were investigated by using qRT-PCR (Figures 7, 8).

Meanwhile the expression levels of miR156 in controls were sharply increased at 1 day and decreased to the minimum at 4 days, and increased again to the maximum at 6 days.

Interestingly, our results found that the expression levels of mdm-miR156, mdm-miR828 and mdm-miR858 were different when fruits were debagged.

” However, the change trends of miR156 and its target gene were rather similar in “Starkrimson.

SPL (squamosa promoter -binding protein-like) transcription factors were predicted to act as the target genes of miR156.

In Arabidopsis stems, the increase of miR156 activity represses the functions of its target gene, SPL (squamosa promoter binding protein-like compounds), and promotes accumulation of anthocyanin, whereas the reduction of miR156 activity leads to the accumulation of flavonols (Gou et al., 2011).

It has been reported that miR156 can positively regulate anthocyanin biosynthesis by SPL transcription factor, and SPL transcription factor can negatively regulate accumulation of anthocyanin through destabilization of a MYB-bHLH-WD40 complex (Gou et al., 2011).

For instance, in miR156 family, the read numbers of mdm-miR156a were 928, while the read numbers of mdm-miR156q were only 6. This large discrepancy of reads among miRNA family members might reflect their divergence of potential physiological roles during fruit development.

The largest miRNA family, miR156, had 29 members, followed by miR171 and miR172 with both 15 members (Figure 3).

The transcription levels of mdm-miR156 in “Granny Smith” after debagging were very low at 0 day, and then continuously increased to the maximum at 2 days, followed by a prominent decrease till 6 days.

” Therefore, the results showed that mdm-miR156 could promote the accumulation of anthocyanin by MdSPL transcription factors in “Granny Smith” after debagging, but not in “Starkrimson.

As for mdm-miR156 in “Granny Smith,” and miR5072 in “Starkrimson,” they can increase anthocyanin concentration upon bag removal.

5

Gleave et al. [55] previously reported putative orthologues of SPL encoding genes as apple miR156 targets, an apple ARF16 as a miR167 target, orthologues of AP2 and TOE1 as miR172 targets and a copper superoxid dismutase, MdCSD as a miR398 target.

Conservation status miRNA family Arabidopsis Oryza(rice) Populus(poplar) Predicted target gene(s) miR156 √ √ √Squamosa promoter -binding proteins[57] miR159/319 √ √ √GAMYB transcription factors[57] miR160 √ √ √Auxin response factors (ARF) [57] miR162 √ √ √DICER-LIKE 1 (DCL1) [57] miR164 √ √ √NAC domain transcription factors[57] miR156/166 √ √ √HD-ZIP transcription factors[57] miR167 √ √ √Auxin response factors (ARF) [57] miR168 √ √ √ARGONAUTE 1 (AGO1) [57] miR169 √ √ √HAP2-like transcription factors[57] miR171 √ √ √Scarecrow-like transcription factors[57] miR172 √ √ √APETALA 2 transcription factors[58] miR390 √ √ √TAS3[59] miR393 √ √ √F-box transcription factors (TIR1) [60] miR394 √ √ √F-box transcription factors[60] miR396 √ √ √GRF, rhodenase[60] miR397 √ √ √laccase[60] miR398 √ √ √Copper superoxid dismutase, CytC oxidase[60] miR403 √ √ √ARGONAUTE 2 (AGO2)[20] miR408 √ √Peptide chain release factor, laccase[20] miR475 √PPR proteins[8] miR476 √PPR proteins[8] Figure 1 Differential expression of miRNAs in apple tissues.

miR156 and miR159 were expressed in all tissues tested, with miR156 expressed to similar levels in all tissues, while miR159 levels were somewhat lower in xylem than in other tissues tested.

Conservation status miRNA family Arabidopsis Oryza(rice) Populus(poplar) Predicted target gene(s) miR156 √ √ √Squamosa promoter -binding proteins[57] miR159/319 √ √ √GAMYB transcription factors[57] miR160 √ √ √Auxin response factors (ARF) [57] miR162 √ √ √DICER-LIKE 1 (DCL1) [57] miR164 √ √ √NAC domain transcription factors[57] miR156/166 √ √ √HD-ZIP transcription factors[57] miR167 √ √ √Auxin response factors (ARF) [57] miR168 √ √ √ARGONAUTE 1 (AGO1) [57] miR169 √ √ √HAP2-like transcription factors[57] miR171 √ √ √Scarecrow-like transcription factors[57] miR172 √ √ √APETALA 2 transcription factors[58] miR390 √ √ √TAS3[59] miR393 √ √ √F-box transcription factors (TIR1) [60] miR394 √ √ √F-box transcription factors[60] miR396 √ √ √GRF, rhodenase[60] miR397 √ √ √laccase[60] miR398 √ √ √Copper superoxid dismutase, CytC oxidase[60] miR403 √ √ √ARGONAUTE 2 (AGO2)[20] miR408 √ √Peptide chain release factor, laccase[20] miR475 √PPR proteins[8] miR476 √PPR proteins[8] Figure 1 Differential expression of miRNAs in apple tissues.

A, Gel blot analyses of miR156, miR159, miR166, miR167 and miR172 expression.

Accumulation of MdSPL target transcripts varied among tissues and was not obviously related to differential accumulation of miR156, as was the case for miR172 and MdTOE1 (Figure 6C).

RNA gel-blot analysis was used to examine the expression of miR156, miR159, miR166, miR167 and miR172 in shoot apex, leaf and stem tissues.

The relative levels of expression were higher for miR159, miR166 and miR167 than for miR156 and especially miR172, which was barely detectable.

D-F, Spatial expression of miR156, miR167 and miR171 in Arabidopsis inflorescence stem; miR156 and miR167, but not miR171, were detected in Arabidopsis phloem by in situ hybridization using appropriate antisense oligonucleotide probes.

B, Stem-loop RT-PCR analyses of miR156, miR159, miR166, miR167 and miR172 expression.

A-C, Spatial expression of miR156, miR167 and miR171 in apple seedling stem; miR156 and miR167, but not miR171, were detected in apple vascular tissue by in situ hybridization using appropriate antisense oligonucleotide probes.

In particular, accumulation of miR156, miR167, miR169, miR390, and miR398 was more than ten-fold higher in the phloem sap than in the vascular tissue, suggesting a possibility of an active mechanism regulating their presence in the phloem sap.

Both miR156 and miR167 antisense probes produced a strong hybridization signal in the phloem (Figure 2D-E), while no signal was detected with miR171 antisense oligonucleotide probe (Figure 2F), consistent with the RT-PCR results.

Using this approach miR156, miR159, miR160, miR162, miR167, miR169, miR396 and miR398 were clearly detectable; miR172, miR390 and miR393 produced a weak amplification signal; miR166 and miR397 amplification did not produce the expected product, but resulted in a smear not detected in the minus-RT control; miR164, miR168, miR171, miR394, miR403, miR408 and the miRNAs specific to poplar (miR475 and miR476) were not detected (Figure 4).

Antisense oligonucleotides corresponding to miR156 and miR167 but not miR171 produced a hybridization signal in the phloem of apple seedling stems (Figure 2A-C).

6

Ectopic expression of apple miR156 h reduces expression levels of AtSPL9 and AtSPL15, and delays the flowering time in transgenic Arabidopsis (Sun et al., 2013).

miR156 and miR172 could also regulate flowering time in response to vernalization through the opposite expression trends (Bergonzi et al., 2013).

The flowering regulators miR156 expressed stronger in the vegetative buds, while miR172 was abundant in the floral buds (Table S4).

In Arabidopsis, miR156 targets a gene family of 11 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors; whereas miR172 regulates six members of the APETALA2 (AP2) transcription factors.

miR156 has been wi dely reported to targeted SPLs.

In DE-miRNAs analysis, only miR398, miR408, miR159, and miR156 expressed stronger in vegetative bud.

The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis.

However, the difference of miR156 expression between the vegetative and the floral buds was not as significant as the ones during phase transition in apple and other woody plants (Wang J. W. et al., 2011; Xing et al., 2014).

In our results, miR156 was expressed at a higher level in the vegetative buds, suggesting that miR156 could also control vegetative growth.

In both annual mo del plant Arabidopsis and polycarpic perennial crops such as Cardamine flexuosa, miR156, and miR172 were first identified to regulate phase transition, the process that also represents the first time of floral transition during plant life circle.

Thus, miR156 might differentially regulate juvenile phase and vegetative phase by its abundance: higher abundance might be important for juvenile phase, while lower abundance might be important for vegetative growth.

Among them, miR398, miR408, and miR156 are all related with the repression of SPL genes, a key factor in juvenile growth and a positive regulator of flowering process (Wu et al., 2009; Zhou C. M. et al., 2013).

In many previous studies in Arabidopsis and other plants, it has shown that the function of miR156 and miR172 in phase transition also include the repression/promotion of flowering process, respectively.

Notably, mdm-miR398b/c, instead of miR156 or miR172, represents the most significant difference among all the DE-miRNAs (Figure 2B, Table S4).

In addition to miR156 and miR172, numerous DE-miRNAs were also identified to involve in floral transition in apple trees.

Among these families, major malus miRNAs, including miRNA156 (9 members), miRNA171 (14), miRNA172 (14), miRNA167 (10), and miRNA395 (9), were detected (Figure 2A).

Several DE-miRNAs were shown to correlate with SPL genes, such as the vegetative bud-enriched miR156, miR159, miR398, and miR408.

In conclusion, in this study we found that the classic mo del of miR156-miR172 in flowering appears to apply to floral transition of apple trees.

miR156 functions to extend juvenile phase and delay flowering, while miR172 leads to early flowering (Wu et al., 2009; Zhou C. M. et al., 2013).

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Moreover, over-expressed miR156 directly prevent the expression of anthocyanin biosynthetic genes (Additional file 5) by targeting SPL9, in Arabidopsis[47].

However, among the target genes, the SPL and R2R3-MYB transcription factors, both of which are known to negatively regulate flavonoid biosynthesis, were experimentally validated to be targets of miR156 and miR159, respectively [46, 47].

The target gene, SBP transcription factor, was validated to be target of miR156/157 families in three Rosa cultivars (Maroussia, Sympathy and Haedang).

We observed that the SBP transcription factor was targeted both by miR156 and miR157 families in ‘Sympathy’, whereas other SBP transcription factors were targeted only by miR156 family in Haedang and ‘Maroussia’ (Figure  5A).

Over -expression of miR156 (Figure  5A) and miR159 (Figure  5B) induced delayed flowering in Arabidopsis by negatively regulating SPL and MYB family transcription factors genes, respectively [60, 61].

miR156 and miR157 in plants have been grouped in one miRNA family due to their high degree of sequence similarity and their conserved target, the SBP transcription factors [22].

In this study, we identified nine miR156 members from all Rosa (Additional file 2), and their target genes, SPL transcription factors, were experimentally validated by 5’ RACE assay (Figure  5).

According to previous studies, miR156, miR159, and miR160 are evolutionary conserved in all land plants, and miR164, and miR172 are conserved in seed-bearing plants [57].

’, ‘Marcia’, ‘Sympathy’, and ‘Vital’, respectively) and its sequencing frequencies were 10 to 100 times more than other relatively abundant miRNA families, including miR156, miR157, and miR167 (Table  4).

8

Temporal regulation of shoot development in Arabidopsis thaliana by miR156 and its target SPL3.

Since then, many researchers have identified miRNAs that silence transcription factors to regulate diverse biological processes in plants; for example, miRNA156 targets members of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors that regulate the transition from vegetative to reproductive growth, tillering/branching, panicle/tassel architecture, and response to abiotic stresses in Arabidopsis (Wu and Poethig, 2006; Wang et al., 2009; Gou et al., 2011; Stief et al., 2014; Wang and Wang, 2015).

Another Md-miR156 paralog, like Md-miR156a (miRBase MIMAT0025867), had a smaller change in relative expression than Md-miR156x, which also targeted a member of the SPL family (Figure 2).

Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156 -targeted SPL transcription factor.

Notably, except for the apple miRNAs/miRNA families mentioned above (Md-miR156, Md-miR164, Md-miR166, Md-miR159, and Md-miR396) that target five transcription factor families, Md-miR395 (miRBase MIMAT0025980) and Md-miR156ab (miRBase MIMAT0025894) exhibited the largest fold change in abundance after ALT1 infection (Figure 2).

Md-miR156 family targets several genes encoding members of the SPL transcription factor family, which are reported to participate in the response to abiotic stress (Stief et al., 2014; Wang and Wang, 2015).

In our libraries of differentially expressed miRNAs, one member of the Md-miR156 family, Md-miR156x (miRBase MIMAT0025890; miRBase website: http://www.

The miR156/SPL module, a regulatory hub and versatile toolbox, gears up crops for enhanced agronomic traits.

miR156-regulated SPL transcription factors define an endogenous flowering pathway in Arabidopsis thaliana.

Arabidopsis miR156 regulates tolerance to recurring environmental stress through SPL transcription factors.

9

However, in our experiment miR156 did not show differential expression between ‘ON’ and ‘OFF’ trees, consistently with the up-regulation of SPL-like genes found in citrus without differential expression of miR156 [88].

Expression of both SPL5 and SPL9 is regulated by the microRNA miR156.

In Arabidopsis, expression of both SPL5 and SPL9 is regulated by the microRNA miR156.

However, in our experiment miR156 did not showed differential expression between the two treatments (Table  2).

Also, two highly conserved microRNAs (miRNAs), miR156 and miR172 [76, 112] target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family transcription factors, which promote the transition from the juvenile to the adult phase, in Populus [110] and key floral repressors belonging to the AP2-like transcription factor family genes [118], respectively.

10

However, miRNA156, miRNA159 and miR172 targeted more than one gene family.

Alignment of multiple sequences and phylogenic analysis of microRNA156 pre-miRNAs in date palm.

B) Phylogenic tree (formed by Neighbor Joining) for miRNA in the miR156 family.

0071435.g003 Figure 3 A) Alignment of twelve pre-miRNA sequences of miR156.

Within the miR156 family, sequence alignments showed that miR156e/j, miR156f/i, miR156a/g, miR156c/h, and miR156k/l had high similarity, and the 12 miRNAs could be divided into five groups based on multiple sequence alignments (Figure 3A–B and Figure S1).

A) Alignment of twelve pre-miRNA sequences of miR156.

As shown in Table 1, the most abundant miRNA family was miR156/157 (12 loci), which has been identified in 45 plant species and which had an average copy number about 18 in the seven other well-studied plant genomes analyzed (Table S2).

C) Blast2 results for two pairs of paralogous date palm contigs containing pre-miR156.

Further sequence comparison between date palm contigs containing miR156 showed that four pairs of miRNAs (miR156e/j, miR156f/i, miR156a/g, and miR156c/h) had highly similar flanking sequences (Table 2).

These thirteen conserved pre-miRNAs belonged to the miR156 (6), miR159 (4), miR160 (2) and miR170 (1) families.

These duplicated regions included miRNAs from all 21 families, and duplication on family miR156 duplicated was detected in all four species.

11

The potential targets of miR858, miR828, and miR156 participate in the fatty acid biosynthetic process.

The juvenile to adult phase transition related microRNAs, mdm-miR156 and mdm-miR172, and the flowering related, mdm-miR535, mdm-miR168, and mdm-miR167, also showed high expression levels in apple shoot tips.

The potential targets of miR166, miR159, and miR156 are involved in meristem phase transition.

The top five miRNA families, mdm-miR156, mdm-miR172, mdm-miR171, mdm-miR167, and mdm-miR399, had more than 10 members.

For example, the mdm-miR156 family had 31 members.

miRNA156 is conserved in embryophytes (Zhang et al., 2006; Taylor et al., 2014) and represents the largest microRNA family in pear (Wu et al., 2014), which like apple, is a member of the Rosaceae.

12

Almost all miRNAs showed, to various degrees, differential expression among the tissues analyzed, with the greatest variation observed for miR156, which was expressed at an abundance of more than 150,000 RPM in root but only 184 RPM in fruit (Figure 1a).

For example, miR156 could target nine members of the squamosa promoter -binding-like protein family, and miR167 targeted six members of the auxin response factor (ARF) family (Table 2; Table S6 in Additional file 1).

The highest read abundance (166,000 RPM) was detected for miR156 and was 5 to 16 times more than other relatively abundant miRNA families, including miR165/166, miR167, miR396, and miR159, whose total abundance ranged from 10,000 to 30,000 RPM (Figure 1a; Table S3 in Additional file 1).

13

This number was soon increased to include miR168 (inhibiting ARGONAUTE1), miR172 (inhibiting APETALA2) (Itaya et al., 2008), and miR156 (targeting CNR) (Zhang et al., 2011).

14

[22] SPLs are a component of the miR156 (micro RNA 156) mediated-age pathway in Arabidopsis, with SPL levels gradually increasing during the transition from the juvenile to adult states.