10 publications mentioning mtr-MIR156j

Open access articles that are associated with the species Medicago truncatula and mention the gene name MIR156j. Click the [+] symbols to view sentences that include the gene name, or the word cloud on the right for a summary.

1

Tissue-specific expression of miR156, miR172, and miR156 -targeted SPL genes in alfalfaTo gain an insight into how miR156 and its target genes are regulated in alfalfa, we evaluated the expression of miR156, its target SPL genes, and miR172 in four tissue types at different developmental time points from the juvenile stage (10 day-old rooted cuttings) to just before flowering.

Our transcriptomic analysis, showed that overexpression of miR156 can affect some similar categories of downstream genes; genes differentially expressed in both genotypes, and which may affect similar phenotypes, but different miR156 expression levels could also affect expression of some unique genes in each genotype, which may affect the degree of phenotypic change relative to WT control.

To gain an insight into how miR156 and its target genes are regulated in alfalfa, we evaluated the expression of miR156, its target SPL genes, and miR172 in four tissue types at different developmental time points from the juvenile stage (10 day-old rooted cuttings) to just before flowering.

Fig. 7Developmental and tissue-specific expression profiles of miR156, miR172, and miR156 -targeted SPL genes in M. sativa.

Genes that are commonly downregulated in both A17 and A11a genotypes include four additional miR156 -targeted SPL genes (MsSPL2, MsSPL3, MsSPL4 and MsSPL9), in addition to the previously reported ones (MsSPL6, MsSPL12 and MsSPL13) [4].

Given the diversity of traits affected by overexpression of miR156 in alfalfa, it is critical to identify and characterize its downstream target genes, especially SPL genes and genes that are regulated by SPLs, as well as understand the functions and behaviours of SPL genes and their target genes by solidly linking each to one or more phenotypes exhibited by miR156OE plants.

In addition, overexpression of AtSPL9 in hyponastic leaves1 mutants - which have lower miR156 expression - caused complete loss of the juvenile phase [52].

This is the first report of changes in global gene expression in response to miR156 overexpression in alfalfa.

The discovered miR156 -targeted SPL genes belonging to different clades indicate miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

Tissue-specific expression of miR156, miR172, and miR156 -targeted SPL genes in alfalfa.

To illustrate changes in global gene expression induced by miR156 overexpression in alfalfa, we conducted (RNA-Seq) on two miR156OE alfalfa genotypes (A11a and A17) generated in our previous study, and which showed reduced plant height and stem thickness; increased branching (main and lateral branches) and node numbers, as well as increased trichome density in leaves [4].

In summary, this is the first report on the effect of miR156 overexpression on global gene expression in alfalfa.

Expression analysis showed that miR156 was primarily expressed in the leaves, with the highest levels observed at the earliest time point, 10 days (Fig.   7a).

The predicted miR156 target sequence (highlighted in yellow) was located in the 3’ untranslated region of (a) MsSPL2, (b) MsSPL3, and (c) MsSPL4 and (d) the open reading frame region of MsSPL9.

The multitude of traits affected in alfalfa by miR156 overexpression could be explained by the fact that its SPL targets, i. e. MsSPL2/3/4, MsSPL6, MsSPL9, MsSPL12 and MsSPL13, belong to clades VI, IV, VIII, V and VII, respectively (Fig.   6a).

A d e novo assembled alfalfa transcriptomeIn order to illustrate the role of miR156 in alfalfa plant development, WT and the two most prominent miR156OE genotypes A11a and A17 [4] were selected for Next Generation Sequencing at the transcriptome level to detect differentially expressed genes (DEGs).

In Lotus japonicus, miR156 -targeted genes, SPLs and WD40, can prolong developmental phase transition, delay flowering time and enhance shoot branching [24].

In addition, three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be downregulated via transcript cleavage by miR156 in alfalfa [4].

MiR156 and its SPL target genes play crucial roles in regulating different aspects of plant growth and development [6– 10].

The Student’s t test was used to analyze the significant differences of each of the tested genes between WT and miR156OE genotypes A11a and A17 (* p < 0.05, ** p < 0.01) In order to illustrate the role of miR156 in alfalfa plant development, WT and the two most prominent miR156OE genotypes A11a and A17 [4] were selected for Next Generation Sequencing at the transcriptome level to detect differentially expressed genes (DEGs).

The height of the letter (amino acid) at each position represents the degree of conservation MsmiR172 is downregulated in miR156OE alfalfa plants MiR156 and miR172 signals are integrated at the SPL3, SPL4, SPL5 and SPL9 genes in the mo del plant Arabidopsis [36, 37].

Among the twenty clones sequenced for each gene, transcript cleavage was detected in all four SPLs outside of their predicted miR156 target sites: 54 bp upstream in nine MsSPL2 clones (Fig.   5a), 42 bp downstream in sixteen MsSPL3 clones (Fig.   5b), 143 bp upstream in eleven clones and 130 bp upstream in one clone of MsSPL4 (Fig.   5c), and 350 bp upstream in seven MsSPL9 clones (Fig.   5d).

Based on the phylogenetic tree analysis, all the current detected SPL genes in the miR156OE plants belong to five different clades, indicating that miR156 plays fundamental and multifunctional roles in regulating alfalfa plant development.

The present analysis investigates whether additional SPL genes are targeted for transcript cleavage by miR156, and what other genes are differentially expressed in miR156OE alfalfa plants.

Under short day conditions, the three genes (AtSPL3/4/5) are negatively regulated by miR156 in an age -dependent manner, and are positively regulated by SOC1 through the GA pathway [36, 46].

In general, MsSPL6 and MsSPL13 had an opposite expression pattern to miR156, with the highest transcript levels observed in the leaves at 40 days (Fig.   7c, e).

These genes are thus potential targets for transcript cleavage by miR156 in alfalfa.

Novel SPL targets of miR156 in alfalfa.

b Length distribution of de novo assembled transcripts As miR156 overexpression affects a number of traits in alfalfa plants [4], we set out to identify DEGs that may be responsible for such traits.

However, unlike findings in Arabidopsis [13], cleavage sites for MsSPL2/3/4 and MsSPL9 in alfalfa were not detected within the predicted miR156 target region.

The Student’s t test was used to analyze the significant differences of each of the tested genes between WT and miR156OE genotypes A11a and A17 (* p < 0.05, ** p < 0.01) We previously generated six alfalfa genotypes (A16, A8a, A8, A11, A17 and A11a) with increased miR156 expression [4].

Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group.

Four additional SPLs (MsSPL2/3/4 and MsSPL9) were discovered to be targeted for silencing by miR156 in alfalfa.

Recently, we overexpressed a precursor of miRNA156 (MsmiR156) in alfalfa, and this led to up to a 2-fold increase in biomass yield, delayed flowering time, enhanced cellulose content and reduced lignin, producing an overall improvement in biomass quality [4].

Fig. 4Validation of the miR156 targeted SPL genes in miR156OE plants using qRT-PCR.

Clades I, II and III represent SPL genes that are not targeted by miR156 (not highlighted with colour in Fig.   6a), while genes from clades IV, V, VI, VII and VIII can undergo cleavage by miR156.

Among the differentially expressed SPL genes, we hypothesize that MsSPL2/3/4 (clade VI) may perform similar functions as AtSPL3/4/5 in Arabidopsis (Fig.   6a), because both of these two groups of SPLs are relatively small in size (420–550 bp) and contain complementary sequences of miR156 in the 3’ UTR.

Many of these GO terms could reflect some traits affected by miR156 overexpression.

Therefore, it appears that diverse levels of miR156 expression may affect alfalfa traits differently.

These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana.

MsSPL9, discovered in this study, to be regulated by miR156 in alfalfa, belongs to clade VIII.

The student t test was used to analyze the significant differences of each tested gene between WT and miR156OE genotypes A11 and A17 (* p < 0.05, ** p < 0.01) To investigate whether miR156 directly targets the four newly discovered SPL genes, we identified their predicted miR156 recognition sites using sequence alignment and used a modified 5’-RACE technique [4] to test for transcript cleavage.

In Arabidopsis, miR156 regulates 10 out of 16 Arabidopsis SPL genes that belong to the same clades as those silenced by miR156 in alfalfa [13, 39].

The student t test was used to analyze the significant differences of each tested gene between WT and miR156OE genotypes A11 and A17 (* p < 0.05, ** p < 0.01)To investigate whether miR156 directly targets the four newly discovered SPL genes, we identified their predicted miR156 recognition sites using sequence alignment and used a modified 5’-RACE technique [4] to test for transcript cleavage.

Two of the genotypes, A17 and A11a (with a 1600- and 3400-fold increase in miR156, respectively), were chosen for RNA-Seq analysis.

The transcript levels of miR156 and miR172 were analyzed by stem loop qRT-PCR [58] and SPL genes by normal qRT-PCR using a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad).

Fig. 5Validation of the miR156 cleavage sites in MsSPL2/3/4 and MsSPL9 transcripts and prediction of the Nuclear Localization Signal.

Detection of miR156 cleavage sites in MsSPL2, MsSPL3, MsSPL4, and MsSPL9 transcriptsThe cleavage sites in alfalfa SPL genes were detected using a modified 5’ rapid amplification of cDNA end (5’-RACE) as previously reported [62].

The height of the letter (amino acid) at each position represents the degree of conservation MiR156 and miR172 signals are integrated at the SPL3, SPL4, SPL5 and SPL9 genes in the mo del plant Arabidopsis [36, 37].

It will be crucial to validate the functions of each SPL gene belonging to different clades to more fully understand the functions of miR156 in determining alfalfa traits.

To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq.

Relative gene transcript levels of (a) miR156, (b) miR172, (c) MsSPL6, (d) MsSPL12, (e) MsSPL13, (f) MsSPL2, (g) MsSPL3, (h) MsSPL4 and (i) MsSPL9 were analyzed by the 2 [-∆CT] method.

This is consistent with our results, which showed relatively high SPL transcript levels were detected in roots where miR156 transcript was undetectable.

A recent publication reported that AtSPL3, AtSPL9 and AtSPL10 were involved in the repression of lateral root growth, and that miR156/SPLs module participates in lateral root primordia progression [55].

In addition, repression of AtSPL2 and AtSPL11 by miR156 is also required for heat stress memory [12].

Detection of miR156 cleavage sites in MsSPL2, MsSPL3, MsSPL4, and MsSPL9 transcripts.

2

For instance, in M. truncatula, a 20 nt variant of miR156 was able to cleave a novel WD40 target in addition to the conserved Squamosa-Binding Protein TF targets (Naya et al., 2010).

Cleavage of a non-conserved target by a specific miR156 isoform in root apexes of Medicago truncatula.

Among PHAS loci which contained two 22 nt miRNA binding sites, those authors found an APETALA2 (AP2)-like gene, which possess one cleavage site for miR172 and a predicted non-cleavable miR156 target site, resembling the two miR390 complementary motives in TAS3.

Thus, the acquisition of the second (miR156) miRNA binding site in MtAP2 certainly happened recently in evolution (Zhai et al., 2011) and this may have consequences on the generation of secondary siRNAs.

Most of them (from miR156 to miR399) corresponded to the set of 21 conserved miRNAs found in nearly all angiosperms (Sunkar and Jagadeeswaran, 2008).

3

Members of the miR156 family target the SQUAMOSA promoter binding like (SPL) TFs [66, 67].

For example, Naya et al. [48] showed that, in addition to the well conserved squamosa binding protein targets, a novel mtr-miR156 isoform was able to cleave a transcript coding for a WD40-like protein in M. truncatula root tips.

These miRNAs fitted into 78 families, for example, 21 'conserved' (miR156 to miR530) and 57 others (miR1507 to miR7696).

In fact, few families contained more than five variants and the greatest complexity was found in three conserved families (Additional file 6: Figure S2; Additional file 2: Table S2a), miR156 and miR169 (6 variants each) as well as miR171 (7 variants).

Isoforms of miR156/157 have been found in root apexes and were less abundant in nodules ([48] and this study), which may lead to differential accumulation of the SPL TFs.

4

We found homologs of known miRNA target genes for several conserved M. truncatula miRNAs, such as SBP for miR156, NAC for miR160, AGO1 for miR168, bZIP for miR165, GRAS for miR171, AP2 for miR172 and low affinity sulphur transporter for miR395.

We analysed the number of reads for conserved miRNAs and miR156, 159 and 166 were represented most frequently in the library (Table 2).

5

These include the positive factors PIF4 and CIB1, bHLH proteins involved in light signaling, and SPL3, which is a target of the miRNA156 pathway controlling juvenility (Liu et al., 2008b; Kim et al., 2012; Kumar et al., 2012).

The microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 module regulates ambient temperature-responsive flowering via FLOWERING LOCUS T in Arabidopsis.

6

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

The microrNA156-SQUAMOSA promoter binding protein-like3 module regulates ambient temperature-responsive flowering via flowering locus in Arabidopsis.

7

Overexpression of miR156 conferred salt tolerance in alfalfa, but plant height and flowering time were negatively affected in the transgenics (Arshad et al., 2017).

8

The reads number for these known miRNAs also varied to a large extent ranging from 1 to 363294, with miR166, miR156, and miR168 families having the most abundant reads in the two libraries.

Five miRNA families (miR399, miR156, miR166, miR171, and miR172) had more than 10 members, and miR156 family, the largest family, had 23 members.

9

Encouragingly, minor perturbations to the abundance of three of the six quantified miRNAs, miR156, miR163 and miR168, were detected in GmDrb2ab leaves (Figure  S12b).

10

Of these families, the most abundant two reads were miR156 and miR166.