Introduction

Vigorous and deep rooting systems are advantageous in plant species when dealing with soil erosion (Reubens et al. 2007), nutrient acquisition, and improving water use efficiency (Tron et al. 2015; Wallace 2000). Effective rooting systems are also important in root crops (cassava, yam, sweet potato, etc.) (Love et al. 2009; Yu et al. 2015b) and plant species such as sheanut in which asexual reproduction is widely used to propagate the plants (Amissah et al. 2013).

Alfalfa is a forage crop with the ability to form a root symbiotic relationship with rhizobia, a process whereby the plant provides carbohydrates in exchange for reduced atmospheric nitrogen from the microorganism. The symbiotic process in legumes has been widely studied elsewhere (Ferguson et al. 2010; Oldroyd and Downie 2008; Oldroyd et al. 2011). Plant exudates (e.g. phenolic compounds) initiate nodule formation in the roots, whereby phenoplics in the rhizosphere attract bacteria to the root and activate the expression of early nodulation (ENOD) genes, and secrete lipo-chito-oligosaccharides, known as nod factors (NF) (Aung et al. 2015b; Ferguson et al. 2010; Oldroyd et al. 2011). Attachment of rhizobia also induces root hair deformations, which allow the bacteria to enter to the root. The growth of bacteria then generates pressure and pushes against the root hair turgor pressure, leading to the formation of infection thread in the extracellular matrix. The infection thread then spreads, and the bacteria are released in the host cell cytoplasm via an endocytotic-type mechanism that encapsulates the bacteria within the plant membrane. The bacteria continue to grow within the host cells and differentiate into bacteroids to fix nitrogen (Gallego-Giraldo et al. 2014; Lamb et al. 2006; Volenec et al. 2002; Wang et al. 2015).

To date, most alfalfa improvement programs have focused on the enhancement of forage yield, forage quality, and stress resistance (Aung et al. 2015b; Lamb et al. 2006; Volenec et al. 2002), and efforts towards improving the root system and below-ground plant development are still rare. Recently, Gallego-Giraldo et al. (2014) demonstrated that modifying lignin by downregulating hydroxycinnamoyl coenzyme A: shikimate hydroxycinnamoyl transferase (HCT) improved forage digestibility and increased nodule numbers in alfalfa. However, nitrogenase activity remained unchanged in these new alfalfa plants and the modification reduced root growth and biomass in alfalfa (Gallego-Giraldo et al. 2014), suggesting that lignin modification negatively affects root development and has a limited potential to promote nitrogen fixation activity.

MicroRNAs (miRNAs) are an emerging class of small RNAs regulating growth and development throughout the entire plant life cycle. MiRNAs have been shown to regulate root development (Wang et al. 2015) and phytohormone accumulation (auxin, cytokinin, abscisic acid, gibberellic acid, ethylene and jasmonic acid) in plants (Curaba et al. 2014), with variable impact from overexpression that is species-specific and miRNA-specific (Aung et al. 2015a; Wang et al. 2015). Plant pri-miRNAs contain short sequences that encode regulatory peptides (Lauressergues et al. 2015). In the case of miR171b and miR165a, introduction of their synthesized peptides to plants specifically stimulated the accumulation of miR171b (leading to reduction of lateral root development) and miR165a (leading to enhancement of main root growth) (Lauressergues et al. 2015). Xie et al. (2012) observed that transgenic rice plants overexpressing miR156 produced more roots, but the roots were smaller compared with unmodified rice. Enhancing miR156 expression also promoted lateral root development in Arabidopsis (Yu et al. 2015a). These authors demonstrated that miR156-targeted SPLs (SPL3, SPL9 and SPL10) are involved in lateral root growth, and are responsive to auxin signaling; pointing to the role of miR156/SPL modules in phytohormone accumulation and lateral root development.

Apart from root development, miRNAs have been reported to influence root symbiosis and nodule number in a complex fashion dependent on the miRNA (Bazin et al. 2012). Li et al. (2010) demonstrated that regulation of genes encoding GSK3-like protein kinase (a regulator of plant immunity) protein by over (mis)expression of miR482 increased the numbers of nodules in soybean. In addition, overexpression of miR172 increased the expression of symbiotic leghemoglobin, and nonsymbiotic hemoglobin, and enhanced nodulation in soybean. However, the expression of miR172 and its positive impact on symbiosis could be downregulated by miR156 overexpression (Yan et al. 2013). Moreover, overexpression of LjmiR156 reduced nodulation and downregulated the transcript levels for nodulation-related genes in Lotus japonicus, whereas overexpression of homologous MsmiR156 increased root length and nodulation in some transgenic alfalfa genotypes (Aung et al. 2015b). These studies imply that the negative impact of miR156 enhancement on root development and nodulation is not universal, but rather species-specific.

In this report, we hypothesized that overexpression of MsmiR156 would have an impact on the entire root system (not just on nodulation and root length), as well as on nitrogen fixation, in alfalfa. We first studied the effects of MsmiR156 on root regeneration capacity and nitrogen fixation activity in alfalfa genotypes with different levels of miR156 transcription and analyzed differential gene expression related to these phenotypes. To understand the impact of MsmiR156 overexpression on global gene expression in alfalfa roots, we also evaluated RNA from the roots of two alfalfa genotypes (an untransformed parental genotype with low MsmiR156 expression versus a transgenic genotype with high MsmiR156 expression) by subjecting them to next Next-Generation RNA Sequencing.

Materials and methods

Plants and growth conditions

Alfalfa plants, including a wild type (WT) parental genotype generated through tissue culture and transgenic genotypes overexpressing MsmiR156 as described in (Aung et al. 2015b), were grown in soil media (Pro-Mix®, Mycorrhizae™, Premier Horticulture Inc., Woodstock, ON, Canada) and maintained in a greenhouse with conditions of 21–23 °C, relative humidity (RH) 70%, 16 h light/day, light intensity of 380–450 watts/m2.

Alfalfa rooting and experimental growth design

To obtain uniform root growth at the same developmental stage, T0 transgenic alfalfa plants (four replicates per genotype) were cut back and shoots were allowed to re-grow three times for 2 months (per cycle). Then, 5-week-old shoots developed after the 4th cutting were used for rooting experiments. Alfalfa stem cuttings (vegetative propagules) (~5 cm long containing three nodes) derived from these shoots were inserted into moistened CleanStart root cubes (Oasis Growing Medium, Kent, Ohio) in plastic trays. A total of three biological replicates (20 propagules per replicate per genotype) were used to test root generating capacity in the root cubes. Three biological replicates (three propagules per replicate per genotype) were rooted to determine root biomass and the number of main roots generated from the stem propagules. At least five biological replicates (five propagules per replicate) were rooted for nitrogenase activity (acetylene reduction). Three-week-old rooted stems were transferred to growth media and inoculated with Sinorrhizobium meliloti for the acetylene reduction assay. Three biological replicates (1 stem propagule per rep per genotype) were used for RNA extraction for qRT-PCR expression analysis. Cuttings were then covered with propagation domes (Ontario Grower’s Supply, London, ON, Canada) and grown in the greenhouse under conditions described above and with routine randomization of position on the growth bench.

Phenotypic analysis of root and nodule development

Root development from the stems was monitored at 12 days after initiation of vegetative propagules. Number of main roots generated from the stem cuttings and root biomass were measured in 3-week-old roots. To measure root biomass, 3-week-old rooted stems were transplanted into plastic pots filled with moistened media (vermiculite mixed with sand), grown for 4 weeks, then harvested and dried at 65 °C for 1 week and dry weight measured.

Nodule numbers and root phenotypes were determined at 3 weeks after inoculation (as detailed below). Phenotypes were photographed using a digital camera (Canon, EOS 300D) and a stereomicroscope (Nikon SMZ1500, Japan).

Inoculation and nitrogenase (acetylene reduction) assay

To determine the effect of MsmiR156 overexpression on nodulation and nitrogen fixation activity, 3-week-old rooted stem propagules were transplanted into round plastic pots (4″) (three stems per pot) containing sterilized solid growth media(Vermiculite, Thermo-O-Rock East, Inc., New Eagle, Pennsylvania, USA) without supplementation of nitrogen or any other fertilizers. For each transgenic alfalfa genotype, eight biological replicates (three rooted stems per replicate) were inoculated with Sinorrhizobium meliloti or water. The rooted stems were inoculated by spreading 1 ml of S. meliloti (strain Sm1021) culture (OD600 ~1.5) or MilliQ water (non-inoculated control) evenly over the soil of each pot with a pipette; then pots were covered with propagation domes as described in Aung et al. (2015b) and allowed to grow for an additional 3 weeks. To eliminate potential microbial contamination, plastic pots, trays and propagation domes were pre-bleached using sodium hypochlorite, and soil media (Vermiculite, Thermo-O-Rock East, Inc., New Eagle, PA, USA) and water were sterilized by autoclaving at 120 °C for 1.5 h.

At 3 weeks after inoculation, inoculated roots were harvested by rinsing off the growth medium with water. To conduct a nitrogenase assay, the amount of ethylene evolution from acetylene was measured on healthy-appearing roots harvested from five randomly selected biological replicates per genotype. The entire root system from each plant was harvested and immediately put into a 40 ml glass vial with a tightly closed lid. Air (20 μl) was then removed from the vial and an equivalent volume of acetylene gas (substrate) was injected gas-tight syringes (Hamilton, Reno NV USA) to create an acetylene-enriched atmosphere in the vial. The sealed vial was incubated for 1 h, and then aliquots (20 µl) of headspace volatiles were withdrawn and injected using a splitless mode onto a column (30 m × 0.32 mm, Agilent J&W GC columns). Volatiles were separated using a Hewlett Packard 5890 Series II gas chromatograph (GC) (Agilent Technologies) with flame ionization detection (FID) to record the retention time of the ethylene peak (indicative of nitrogenase activity). (The GC inlet was maintained at 250 °C and 20 psi, the column oven at 90 °C, and the FID at 260 °C). To generate a calibration curve and to determine a retention time, ethylene gas standards (20 µl) (Air Liquide America Specialty Gases LLC, Plumsteadville, PA, USA) were also sampled from the headspace of standards vials and manually injected into the GC at a concentration of 50, 100, 500 and 1000 ppm. The amount of ethylene released from acetylene reduction was then calculated and expressed as root nmol/plant.

RNA extraction

To extract RNA from stem cuttings, the stem was homogenized to a very fine powder in liquid nitrogen using a mortar and pestle. Total RNA was then isolated using an RNeasy Plant Mini Kit (Qiagen, Mississauga, ON, Canada) as described in the instruction manual. RNA was also isolated from 3-week-old roots, 1-month-old roots, and 6-week-old roots following 3 weeks of incubation after inoculation with S. meliloti or sterilized MillQ water as described previously (Aung et al. 2015b).

Next generation sequencing

RNA extracted from 1-month old alfalfa roots of the wild type (WT) parental genotype and transgenic genotype A17 was analyzed for quality using a Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies, Waldron, Germany). High quality RNA (one independent RNA extraction per biological replicate from one stem propagule) was subjected to Next Generation Sequencing (NGS) as a fee-for-service at The Centre for Applied Genomics (SickKids® Hospital, Toronto, ON, Canada). Four independent RNA libraries (i.e. four biological replicates) were prepared per genotype using 500 ng total RNA enriched for polyA RNA, then fragmented for 4 min at 94 °C, followed by double stranded cDNA synthesis, end-repair, 3′-adenylation, ligation with specific multiplex adapters, and cDNA library amplification as described in an Illumina TruSeq mRNA Library preparation protocol by Illumina. RNA library size was determined using a Bioanalyzer 2100 DNA High Sensitivity chip (Agilent Technologies) and quantified by qRT-PCR using the Kapa Library Quantification Illumina/ABI Prism Kit protocol (KAPA Biosystems). Libraries were then pooled in equimolar quantities for multiplex paired-end sequencing using an Illumina HiSeq 2500 platform (six samples per lane) using a Rapid Run Mode flow cell as recommended by the manufacturer. Initial base calling and quality filtering of Illumina HiSeq 2500 image data was performed using the default parameters of the Illumina HiSeq 2500 Pipeline GERALD stage. The data quality was checked using Illumina’s run viewer software which allows real-time monitoring as the sequencing runs progress. trimAll implemented in Biocluster was used to remove adaptor sequences and to trim bases with quality lower than 30 (phred 33 quality scores). Raw RNA-SEQ data were submitted to www.ncbi.nlm.nih.gov (Accession Number SRR2153020).

NGS data were analyzed using TopHat v.2.1.0 (http://tophat.cbcb.umd.edu/) (Trapnell et al. 2009). Transcript assembly, final transcriptome assemblies, quantification of mapped reads, extraction of differential expression values (Fragment Per Kilobase of transcript per Million fragments mapped), and data normalization were carried out using Cufflinks, Cuffcompare, Cuffmerge, and Cuffdiff in the Cufflinks tool suite (version 2.2.1) (http://cufflinks.cbcb.umd.edu/) (Trapnell et al. 2010) as described in Trapnell et al. (2012). Statistical analysis and visualization of the RNA-seq data was performed in R with a CummeRbund package in Cufflinks (Trapnell et al. 2012). RNA seq data were expressed as Fragments Per Kilobase of transcript per Million fragments mapped (FPKM) and a ratio of A17:WT. M. truncatula gene IDs were then used to search for gene ontology (GO) identifiers using agriGO, a GO analysis toolkit for the agricultural community (http://bioinfo.cau.edu.cn/agriGO/) (Du et al. 2010). Gene ontology identifiers were then analyzed, summarized into categories and sub-categories, and common descriptors of the function were determined using Reduced Visualization Gene Ontology (REVIGO) software (http://revigo.irb.hr/) as described in Supek et al. (2011).

Quantitative real-time PCR

Total RNA (1 μg) isolated from the corresponding tissues was used to synthesize cDNA using qScript™ cDNA SuperMix (Quanta Bioscience, Mississauga, ON, Canada) following the manufacturer’s instructions and guidelines for real-time PCR experiments. PCR reactions were prepared and the transcript levels were monitored as in (Aung et al. 2015b) using primers listed in Table S1.

Statistical analysis

Raw NGS root data were analyzed by R test statistics in Cufflinks software (Trapnell et al. 2010) on four biological replicates per genotype (described above). Student t tests implemented in GraphPad (Prism 6) were used to conduct statistical analysis for qRT-PCR and morphological data using three biological replicates (described above). Significant differences of the means were determined between MsmiR156 overexpression plants and the wild type (WT) parental genotype (control) or between S. meliloti-inoculated or water-inoculated (control) plants.

Results

Effects of MsmiR156 overexpression (OE) on root regenerative capacity in alfalfa

Transgenic alfalfa plants and wildtype (WT) alfalfa were vegetatively propagated by stem cuttings from six MsMIR156OE genotypes and their parental non-transgenic genotype to observe root regeneration capacity. Root regeneration from stem nodes could be detected in one or more of the MsMIR156OE genotypes as early as 12 days after vegetative propagation, although root regenerative capacity was unequal among the transgenic plants overexpressing MsmiR156 (Fig. 1a–d). The number of rooted stem propagules (out of a total of 20 evaluated per rep) was significantly higher in transgenic MsMIR156OE alfalfa genotypes compared with the WT control (Fig. 1a, b). Increase in root regenerative capacity was observed earliest in genotype A17. Genotypes A8a, A11a and A17 also had a higher number of adventitious roots regenerated from the stems (Fig. 1c).This enhanced root regenerative capacity may be limited to very early root development after stem cutting (though we did not test regeneration of roots after root wounding), and MsMIR156OE plants and WT plants may additionally have unequal root growth profiles at different stages of root development. For example, a significant increase in root biomass was not observed in the young MsMIR156OE alfalfa rooted cuttings by 3 weeks after planting the stem cuttings (potentially due to high variability) (Fig. 1d), while roots of MsMIR156OE plants were longer than WT roots at a more mature stage.

Fig. 1
figure 1

Effect of MsmiR156 overexpression on root development in alfalfa. a Typical root regeneration phenotype from stem cuttings at 12 days after vegetative propagation (bar scales 20 mm). b Number of rooting stems arising from 20 stems (per rep) at 12 days after vegetative propagation. c Number of adventitious roots generated from stem cuttings at 3 weeks after propagation. d Root biomass (dry weight) in 3-week old roots. Main roots and root biomass in b and c were measured from three plants per biological replicate. Values are mean ± SE (n = 3). One or two asterisks indicates significant differences of the means compared with a wild type parental control at P ≤ 0.1 or 0.05, respectively (t test)

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Analysis of nitrogen fixation activity in alfalfa

To determine the effect of MsmiR156 on alfalfa nitrogen fixation activity, we analyzed the impact of MsMIR156OE on nodulation, nitrogenase activity, and the ability of our transgenic plants to thrive in the absence of N fertilizer. Young 6-week-old transgenic alfalfa overexpressing MsmiR156 enhanced nodulation by Sinorrhizobium meliloti when observed 3 weeks after inoculation (Fig. 2a). In particular, a significant increase in nodulation was observed in genotype A8a, A11, A11a and A17 though high variation also obscured this trend in other OE genotypes (Table S2). During this time, shoots and branches in wild type control (inoculated) plants displayed nitrogen deficiency symptoms (yellow leaves with no new shoots) compared with healthy larger green shoots in MsMIR156OE plants inoculated with S. meliloti, though no significant difference in shoot biomass was observed even at a later date, i.e., at 4-week after inoculation (Fig. 2b; Table S2).

Fig. 2
figure 2

Phenotypes of MsmiR156 overexpression genotypes of 6-week-old rooted stems. a Nodulated root phenotypes at 3-week after inoculation with S. meliloti (bar scales 5 mm). b Aerial growth and nitrogen deficiency symptoms at 4-week after inoculation. c Nitrogenase activity (acetylene-derived ethylene evolution) in transgenic alfalfa at 3 weeks after inoculation with Sinorrhizobium meliloti. Each bar represents the average of five biological replicates, where each replicate represents the emitted ethylene from the entire harvested root of one plant. Values were mean ± SD (n = 5) for WT and six MsMIR156OE genotypes. Ethylene was undetectable in negative controls (non-inoculated plants) within the limits set for the assay. d, e, f Transcript levels for S. meliloti d FixK, e NifA, and f RpoH genes in alfalfa roots inoculated with rhizobia. Expression was analyzed using three biological replicates and two technical replicates. Acetyl CoA carboxylase 1 and acetyl CoA carboxylase 2 genes were used as the reference for normalization. One, two or three asterisks indicate significance from wild type control at P < 0.1, 0.05 or 0.01, respectively (t test)

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An acetylene reduction assay measuring nitrogenase activity showed that the nodulated roots of transgenic alfalfa genotypes A8a, A11, A11a and A17 significantly increased ethylene evolution after 1 h incubation of 3-week-old root biomass with acetylene substrate (Fig. 2c, Fig. S1). The amount of ethylene production was the highest from roots of genotype A17 (8.3 ± 1.7 nmol/plant) whereas the control plant contained the lowest level of ethylene (4.3 ± 0.1 nmol/plant). qRT-PCR expression analysis also indicated that MsmiR156 overexpression affected the expression of bacterial genes FixK (providing activation of nodule respiration), NifA (nitrogenase-encoding) and RpoH (sigma 32 factor for effective nodulation) in alfalfa roots inoculated with S. meliloti. Compared with the control plants, the transcript levels of FixK, NifA and RpoH were significantly increased in A8, A8a, A11, A11a and A17 genotypes (Fig. 2d–f, respectively). These results indicate that MsmiR156 overexpression has strong potential to promote nitrogenase and nodulation activity in alfalfa (and to enable alfalfa to grow in the absence of N fertilizer).

Effect of MsmiR156 overexpression on the transcriptome of young alfalfa roots

Since miR156 overexpression enhanced root emergence capacity in some of our transgenic alfalfa genotypes (particularly genotype A17 with the most consistent root phenotype), total RNA from 1-month-old roots of the wild type (WT) parental alfalfa genotype and A17 were subjected to Next Generation Sequencing (NGS) using four RNA libraries per genotype. In total, 180 million reads were generated from these eight RNA libraries. RNA sequences were compiled and analyzed using TopHat and Cufflinks software (Trapnell et al. 2012) and queried against the publicly available M. truncatula genome database (http://medicago.jcvi.org/MTGD/?q=home) to annotate sequence reads. To date, the complete M. sativa genome sequence is still not available, and as such, we characterized our NGS data based on this closely related species.

Differentially expressed genes were identified based on the positive (+) and negative (−) values of log2 fold changes between the frequency of reads (Fragments Per Kilobase of transcript per Million mapped reads) from the A17 roots relative to that of WT parental roots (Table S3).Of the total 8833 differentially expressed transcripts, approximately 1521 (17%) genes have so far been annotated using the publicly available M. truncatula genome database, in which, 982 gene transcripts were downregulated (− values) and 539 transcripts were up-regulated (+ values) in A17 roots relative to WT tissue (Table S3). Based on reduced visualization gene ontology (REVIGO, (Supek et al. 2011)) analysis, which summarizes Gene Ontology (GO) terms, we noticed that mainly genes involved in a few broad functional categories (biological, metabolic processes, transport, oxidation–reduction processes, and regulation of transcription, as well as a few others) were downregulated, whereas those involved in molecular function, catalytic activity, nucleic acid binding, hydrolase activity, oxidoreductase activity, ATP binding, and a few others) were up-regulated (Fig. 3a, b).

Fig. 3
figure 3

REVIGO output showing gene ontology frequency and functional categories of a up-regulated and b downregulated alfalfa root genes. In total, 982 annotated gene transcripts were downregulated and 539 were up-regulated. REVIGO analysis was conducted using GO terms obtained from identified M. truncatula genes. Note not all M. truncatula identifiers (Gene ID) are currently available. Next generation sequencing reveals differential gene expression in 1-month-old roots of MsMIR156OE genotype A17 and WT parental genotype roots. Number of “reads” for c SPL genes; d nodulation genes; e genes involved in root growth and development and f genes involved in phytohormone signalling pathway. αFPKM values (Fragments Per Kilobase of transcripts per Million fragments mapped). Values are mean ± SE (n = 4). Asterisk indicates significance difference of the means between wild type and A17 transgenic alfalfa genotype (P < 0.05). LysM lysin motif, NTR nodulin transporter, ENOD93 early nodulin 93, Cle clavata3/ESR (CLE) gene family member, NR nodulin receptor, Cyclops cyclops, RPT1 root phototropism-like protein 1, RPT2 root phototropism-like protein 2, LRP lateral root primordium, RHD root hair defective, RCE root cap/late embryogenesis, ARF auxin response factor, AUX auxin-responsive AUX/IAA family protein, GR gibberellin receptor GID1, GRF gibberellin-regulated family protein, AOC Allene oxide cyclase, COD cytokinin oxidase/dehydrogenase-like protein, AAR1 abscisic acid receptor PYL9-like protein, AAR2 abscisic acid receptor, ERT ethylene-responsive transcription factor 1B, CRH cytokinin receptor histidine kinase, ERF ethylene response factor

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In addition, REVIGO analysis showed that differentially expressed genes were involved in 132 different functional categories when sorted by gene ontological terms (132 GO terms), which could be grouped into 17 representative larger categories (Tables S3 and S4). The majority of genes were involved in peptidyl-lysine modification to peptidyl-hypusine (44 GO terms), followed by genes involved in response to water stress (15 GO terms), sucrose transport (15 GO terms), and plant cell wall organization (13 GO terms) (Table S4). In addition, REVIGO analysis uncovered a number of differentially expressed genes involved in multiple biological processes, including fructose 2,6-bisphosphate metabolism (10 GO terms), L-arabinose metabolism (6 GO terms), sulfate reduction (5 GO terms), putrescine biosynthesis (4 GO terms), maintenance of fidelity involved in DNA-dependent DNA replication (4 GO terms), steroid metabolism (4 GO terms), regulation of pH (3 GO terms), is citrate metabolism (3 GO terms), sexual reproduction (2 GO terms), and microtubule-based movement, carbon utilization, lignin catabolism and flavonoid biosynthesis (1 GO term each) (Table S4).

Expression analysis of genes involved in nodulation, root growth, and phytohormones during early stages of root development

Crosstalk between phyotohormones and miRNAs affects root growth and development in plant species, and has particularly been documented in Arabidopsis (Curaba et al. 2014). In our study, NGS data showed that miR156-targeted SPLs (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) and genes involved in nodulation, root growth, and phytohormone signalling were differentially expressed in 1-month-old roots of the MsmiR156-enhanced genotype A17 relative to the WT alfalfa genotype (Fig. 3c–f). As previously observed, the transcript levels of SPL6, SPL12 and SPL13 were reduced in A17 genotype compared with those in WT (Fig. 3c). In addition, the levels of nodulation genes LYSIN MOTIF (LysM), NODULIN TRANSPORTER (NTR) and EARLY NODULIN 93 (ENOD93) were increased in A17 roots compared with WT roots, while those of a CLAVATA3/ESR (CLE) gene family member, the NODULIN RECEPTOR (NR) and CYCLOPS were reduced (Fig. 3d). Moreover, the transcript levels of ROOT PHOTOTROPISM-LIKE PROTEIN1 (RPT1), RPT2, and LATERAL ROOT PRIMORDIUM (LRP) were reduced in A17 roots, whereas those of ROOT HAIR DEFECTIVE (RHD) and ROOT CAP/LATE EMBRYOGENESIS (RCE), were increased (Fig. 3e). NGS data analysis also revealed differential expression of the genes involved in the biosynthesis of phytohormones in the roots, suggesting that MsmiR156 overexpression modulates global gene expression to coordinate (and promote) root development in alfalfa (Fig. 3f).

Based on the NGS data, we compared the effects of MsmiR156 overexpression on alfalfa root regenerative capacity and nodule development by evaluating qRT-PCR transcript profiles of several genes expressed in early stages of root development (Fig. 4a–h). Among the transgenic alfalfa genotypes, A17 contained the highest abundance of root MsmiR156 transcripts whereas the wild type control expressed the lowest level of root MsmiR156 (Fig. 4a, b). However, the transcript level of MsmiR156 was inconsistent with the levels of root development genes in different transgenic genotypes. Here, overexpression of MsmiR156 differentially regulated the expression of ROOT CAP/LATE EMBRYOGENESIS (RCE), ROOT PHOTOTROPISM-LIKE PROTEIN 2 (RPT2) and ROOT HAIR DEFECTIVE (RHD) genes in 12-day old rooted stems and/or 3-week old alfalfa roots, while the transcript level of MsmiR156 was similar within any one genotype between these two growth stages (Fig. 4). For example, RCE-related genes were significantly increased in A8, A8a, A11a and A17 genotype rooted stems and not in A11 genotype (Fig. 4c). Moreover, significant effects of MsmiR156 overexpression on RPT2 gene transcript level was observed in A8a in 12-day old rooted stem, and A11, A11a and A17 genotypes in 3-week old roots (Fig. 4e, f).

Fig. 4
figure 4

Effect of MsmiR156 overexpression on root growth-related alfalfa gene expression monitored by qRT-PCR. Gene expression in a, c, e, g 12-day-old entire stem cutting propagule and b, d, f, h 3-week-old roots. Expression profiles of a, b MsmiR156; c, d RCE; e, f RPT2, and g, h RHD genes. Expression was analyzed using three biological replicates and two technical (machine) replicates. Acetyl CoA carboxylase 1 and acetyl CoA carboxylase 2 were used as the reference for normalization. One, two or three asterisks indicate significant differences from wild type control at P ≤ 0.1, 0.05 or 0.01, respectively (t test). Abbreviations are mentioned in Fig. 3 legend

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MsmiR156also affected the expression of genes involved in the biosynthesis of phytohormones during the early stages of root development (Fig. 5a–j). At 12 days after vegetative propagation, transgenic alfalfa displayed differential qRT-PCR expression of phytohormone-related genes in rooted stems. An increase in the abundance of MsmiR156 was correlated with a reduction in transcript levels of the AUXIN-RESPONSIVE AUX/IAA FAMILY PROTEIN (AUX) gene in all transgenic genotypes except for genotype A16 compared with the WT genotype (Fig. 5a). Similarly, overexpression of MsmiR156 correlated negatively with the transcript levels of AUXIN RESPONSE FACTOR (ARF), and GIBBERELLIN-REGULATED FAMILY PROTEIN (GRF), GIBBERELLIN RECEPTOR GID1(GR) ABSCISIC ACID RECEPTOR 2 (AAR2) and CYTOKININ RECEPTOR HISTIDINE KINASE (CRH) genes in a number of the transgenic alfalfa genotypes (Fig. 5b–f). Interestingly, an increase in the transcript levels of MsmiR156 positively correlated with the expression of CYTOKININ OXIDASE/DEHYDROGENASE-LIKE PROTEIN(COD) and ALLENE OXIDE CYCLASE(AOC) in transgenic alfalfa genotypes, and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR 1B(ERT) (in A8a, A11, A11a and A17) and ETHYLENE RESPONSE FACTOR(ERF) (in A17) genes in 12-day-old rooted stems (Fig. 5g–j). In 3-week-old roots, however, significant changes (increases) in phytohormone-related gene expression only occurred for a few transgenic alfalfa genotypes (AUX, GRF, COD, and ERT) (Fig. S2a–j). These results indicate that MsmiR156 regulates the expression of genes involved in the biosynthesis of phytohormones and modulates root development mainly during the early stage of rooting development(i.e., in 12-day-old stem cuttings), although we did not test expression profiles at a much later stage when MsMIR156OE plants showed a longer root phenotype compared with WT plants.

Fig. 5
figure 5

Effect of MsmiR156 overexpression on phytohormone-related gene expression in 12-day-old entire stem cuttings monitored by qRT-PCR. Expression profiles of a AUX, b ARF, c GRF, d GR, e AAR2, f CRH, g COD, h AOC, i ERT, and j ERF genes. Expression was analyzed using three biological replicates and two technical (machine) replicates. Acetyl CoA carboxylase 1 and acetyl CoA carboxylase 2 gene were used as the reference for normalization. One, two or three asterisks indicate significant differences from wild type control at P < 0.1, 0.05 or 0.01, respectively (t test). Abbreviations are indicated in Fig. 3 legend

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Analysis of qRT-PCR transcript levels for nodulation genes showed that MsmiR156 overexpression activated the expression of several nodulation genes in root primordia of 12-day-old stem cuttings and 3-week-old roots for several-to-most of the transgenic alfalfa genotypes (Fig. 3S). Increase in miR156 abundance correlated with increased transcript levels for CLE (A8, A8a, A11, A11a and A17) (Fig. S3a), ENOD93 (A11a) (Fig. S3c), LysM (A8, A8a, A11, A11a and A17) (Fig. S3e), NR (A16, A8 and A17) (Fig. S3g), and NTR (A11, A11a and A17) (Fig. S3i) at this stage of root development. In addition, a significant decrease in the transcript level of CYCLOPS was also observed in genotype A8a and A11 in 12-day-old stem cuttings (Fig. S3k).

Next, we inoculated 3-week-old rooted stems with Sinorrhizobium meliloti (Sm1021), or sterilized water (as a non-inoculated control) and analyzed the expression of nodulation genes by qRT-PCR in 6-week-old roots (3 weeks after inoculation). Compared with the non-inoculated plants, the transcript levels of CLE and ENOD93 genes were significantly increased in A11, A11a and A17, and decreased in the control roots (Fig. S3b, d). Similarly, the transcript level of the LysM gene was reduced in control and A16 roots, but increased in A11 and A17 genotypes (Fig. S3f). However, enhanced levels of MsmiR156 (as in genotypes A8, A8a, A11, A11a and A17) showed no significant increase in the root expression of NR, NTR and CYCLOPS genes at 3 weeks after inoculation (Fig. S3h, j, l).

Discussion

Crop breeding is aimed mainly at maximizing yield and quality in new cultivars or at protecting crops against environmental or biotic stress, but the root system in crop productivity has been less utilized as a focus for crop improvement (Sarkar et al. 2013). Recently, several strategies have been implemented to assist breeding programs with improving root systems (de Dorlodot et al. 2007; Paez-Garcia et al. 2015; Wasson et al. 2012). Careful selection and crossing of superior germplasm has been proposed for improving the root system and water intake in wheat crops (Wasson et al. 2012). Quantitative trait loci (QTL) mapping for root system architecture has been used in root trait selection (Sarkar et al. 2013; Wasson et al. 2012). Both approaches are laborious and time-consuming. Although progress has been made in alfalfa breeding aimed at improving the above-ground forage (Aung et al. 2015b; Lamb et al. 2006), little attention has been paid to improving the performance of the root system. Lignin modification by downregulating hydroxycinnamoyl co-enzyme A: shikimate hydroxycinnamoyl transferase (HCT) has increased nodule numbers; nevertheless, this new strategy suffers from limited potential since it was unable to increase nitrogenase activity and root biomass in alfalfa (Gallego-Giraldo et al. 2014). Previously, we showed that overexpression of MsmiR156in alfalfa produced a range of new growth phenotypes that included an increase in nodulation, root length, and forage biomass yield when mature plants were examined (Aung et al. 2015b). This suggested that enhancement of MsmiR156 expression had strong potential to be a novel rapid strategy to enhance both root development and nitrogen fixation capacity in alfalfa and led us to examine root traits in our MsMIR156OE genotypes at much younger stages (≤6 weeks after vegetative propagation).

MsmiR156 promotes root development in alfalfa

Overexpression of MsmiR156 enhanced alfalfa root regenerative capacity during vegetative propagation. The enhancement of root emergence was observed as early as 12 days after vegetative propagation was initiated. Compared with WT control plants, the numbers of rooted stems were significantly increased in all MsMIR156OE genotypes except A16. In addition, the numbers of adventitious roots generated from the stem nodes were significantly increased in genotypes (A8a, A11a and A17), which had the highest levels of MsmiR156 transcripts. Recently, a functional study of miR156-SPL regulatory network also showed that miR156-targeted SPL genes inhibit adventitious root development in Arabidopsis (Xu et al. 2016). However, significant improvement in root biomass was not observed during the early stage of root development (3-week-old roots). Instead, overexpression of MsmiR156 increased root length in these alfalfa genotypes later on, as described earlier after the newly established plants matured (Aung et al. 2015b). Gallego-Giraldo et al. (2014) had reported that downregulating HCT significantly reduced root length and root biomass in alfalfa in both greenhouse and field conditions. In contrast, enhanced root regeneration capacity in young MsMIR156OE propagules and the previously demonstrated increase in root length in more mature MsMIR156OE plants suggest that stem cutting propagation is a faster method of enhancing root establishment and root length compared with other methods used in traditional (seed propagation) or molecular marker-assisted plant breeding.

Improvement of alfalfa nitrogen fixation with MsmiR156

Root symbiosis with rhizobial species provides leguminous plants with reduced atmospheric nitrogen in exchange for carbohydrates for the bacteria (Ferguson et al. 2010; Oldroyd and Downie 2008; Oldroyd et al. 2011). MsmiR156 overexpression increased nodule numbers, nitrogenase activity, and the transcript levels of S. meliloti’s RpoH, FixKand nifA genes in young transgenic alfalfa genotypes (A8a, A11, A11a and A17) compared with WT control plants. Moreover, WT plants inoculated with either S. meliloti or water (as a control) in the absence of N fertilizer displayed N deficiency symptoms in aerial plant organs (pale green or chlorotic leaves and shoot branches), whereas MsMIR156OE genotypes inoculated with S. meliloti were green in the absence of N fertilizer.

Previous studies have shown that NifA and FixK induce the transcription of nitrogen fixation genes, such as nifHDKE, fixABCX, fixNOQP and fixGHIS operons (Fischer 1994; Mitsui et al. 2004), and that S. meliloti’sRpoH is required for effective nitrogen fixation in alfalfa (Mitsui et al. 2004). An increase in transcript levels of CLE, ENOD93 and LysMgenes positively correlated with nodulation in 3-week-old roots (OE genotypes A11 and A11a) inoculated with S. meliloti. Nodulin genes are differentially and sequentially expressed during nodule development (Govers et al. 1986; Scheres et al. 1990). According to Okamoto et al. (2009), LjCLE1 andLjCLE2 repress nodulation in L. japonicus, whereas LjCLE3 has no impact. Moreover, the transcript levels of MtCLE12 and MtCLE13 increase during early nodule development in M. truncatula (Mortier et al. 2010). In L. japonicus, miR156 overexpression reduces the transcript levels of ENOD, SymPK, CYCLOPS and represses the number of nodules in plants inoculated with Mesorhizobium loti (Wang et al. 2015).

Collectively, this data supports our earlier preliminary findings that MsmiR156 enhances nodulation in mature alfalfa (Aung et al. 2015b). It contrasts with the significant reduction in nodulation when LjmiR156 is overexpressed in L. japonicas (Wang et al. 2015) or ectopically in alfalfa (Aung et al. 2015a) and with the failure of HCT silencing to improve alfalfa N fixation even though nodule number was enhanced (Gallego-Giraldo et al. 2014). However, the value of miR156 enhancement as a general tool to improve N fixation in a range of legume species may lie in species-specific gene sources, such as MsmiR156 rather than LjMiR156. Hence, MsmiR156 should now be tested for its impact on a range of heterologous legume species and to determine whether MsmiR156 overexpression impacts negatively on any other crop traits, as occurs with the soybean GmmiR156 (Yan et al. 2013).

Global gene expression profiles in young MsMIR156OE alfalfa roots impacts the hypusine system, root development genes, and phytohormone genes

NGS has been widely used to unravel global gene expression patterns during root growth and development in many plant species (de Vries et al. 2015; Ghorbani et al. 2015; Trevisan et al. 2015; Vayssières et al. 2015; Yin et al. 2015). However, little is known about the effect of miR156 on global gene expression during root development of alfalfa. In this study, we used an NGS approach to investigate the impact of miR156 overexpression on global gene expression in young alfalfa roots in one of our most valued MsMIR156OE genotype A17. In total, 1521 gene transcripts (982 downregulated and 539 up-regulated) were annotated in A17 (using the M. truncatula genome database) as differentially regulated in young roots by MsmiR156 overexpression relative to roots of a WT parental control genotype. The majority represent genes involved in modification of peptidyl-lysine to peptidyl-hypusine. Hypusine is a polyamine-induced amino acid found to replace lysine in the eukaryotic translation initiation factor 5A (eIF5A) (Park 2006). Hypusine-containing eIF-5A appears to produce proteins with stretches of consecutive proline residues (Malekpoor Mansoorkhani et al. 2014) and can be modified by deoxyhypusine synthase and/or deoxyhypusine hydroxylase. Although the effect of hypusine modification is poorly understood in plants, transgenic Arabidopsis plants with constitutively suppressed AteIF5A-2 showed resistance to disease and programmed cell death induced by virulent Pseudomonas syringae pv. tomato DC3000 Pst DC3000 processes (Hopkins et al. 2008). Treatment of Arabidopsis with the hormone abscisic acid also alters the hypusine modification system (Belda-Palazón et al. 2014). Moreover, depletion of genes coding for hypusine modification enzymes causes lethality in adult mice, suggesting that modification of hypusine is critical (at least for mamalian homeostasis) (Dever et al. 2014; Pällmann et al. 2015). Hence, MsmiR156 potentially may also regulate this important translation factor in alfalfa.

Effective root systems are essential to ensure water and nutrient (e.g. nitrogen, phosphorus and iron) uptake for proper growth and development (Malekpoor Mansoorkhani et al. 2014; Ruiz Herrera et al. 2015). Our NGS data revealed a change in transcripts for a number of root genes with potential roles in sucrose transport and water relations (drought, flooding, etc.), implying that miR156 may reprogram several metabolic and signalling pathways in the young alfalfa roots. NGS analysis also showed a global change in the expression of many other genes in alfalfa roots.

At this point, it is still unclear how an alteration in these NGS transcriptome profiles in 1-month-old roots influenced root growth and development, since a number of the transcripts are related to genes that have not been functionally characterized or their roles in several metabolic pathways are not well understood in alfalfa. However, our NGS data revealed expression changes in several genes that are understood to function in root development. RHD and RCE gene transcripts were significantly increased, whereas transcripts encoding RPT2 and LRP were significantly reduced in A17 roots compared with WT alfalfa roots. Consequently, we confirmed the expression of root genes impacting root development and nodulation in emerging roots of 12-day-old stem cuttings and 3-week-old roots by using qRT-PCR analysis. In this case, we also observed that the transcript level of RPT2 was also significantly increased in genotypes A11, A11a and A17 cuttings in 3-week-old roots even though RHD expression levels were not significantly different compared with WT tissues. In maize, LATERAL ROOT PRIMORDIA 1 (LRP1) acts as a transcriptional activator in downstream auxin signalling of the AUX/IAA gene (Zhang et al. 2015). However, Inada et al. (2004) showed that RPT2 and NONPHOTOTROPIC HYPOCOTYL 3 function as signal transducers to regulate hypocotyl phototropism in Arabidopsis. Moreover, RHD was previously shown to be involved in adventitious and lateral root formation in populous (Xu et al. 2012). Unfortunately, our search of the current literature revealed no information on the roles of RCE gene in plants. In the future, it will be important to determine the full nature of root development expression changes in MsMIR156OE alfalfa, since we have only investigated very early vegetatively propagated roots and root length in mature plants and have not yet looked in detail at lateral root formation in alfalfa, nor in seedling root emergence and establishment.

Transition between developmental stages requires interactions of different phytohormone pathways to coordinate organogenesis or tissue outgrowth (Curaba et al. 2014). Manipulation of gene regulatory networks and phytohormone signalling pathways that regulate the nodulation process affect nitrogen fixation activity in plants (Ferguson and Mathesius 2014; Foo and Davies 2011; Gallego-Giraldo et al. 2014; Mohd-Radzman et al. 2013; Yan et al. 2013). In maize, nitrate sensing induces a complex transcriptomic and proteomic reprogramming that affects biosynthesis and signalling of phytohormones in the root apex (Trevisan et al. 2015).

In this study, NGS data showed that expression of auxin, gibberellin and allene oxide cyclase (AOC)-related genes was significantly increased in young roots of genotype A17 compared with WT alfalfa roots, whereas expression of genes with functions related to cytokinin, abscisic acid receptor and ethylene was significantly reduced in A17. Using qRT-PCR, we observed that transcript levels of AUX, and ARF genes were reduced in 12-day-old transgenic alfalfa stem cuttings consistent with an increase in root regenerative capacity in transgenic genotypes at this stage, suggesting that overexpression of MsmiR156 inhibits the auxin signalling pathway in order to enhance root initiation. In rice and Arabidopsis, auxin treatment represses the level of miR156, which suggests that auxin may well inhibit root development through miR156/SPL regulatory networks in these species (Curaba et al. 2014; Liu et al. 2009; Marin et al. 2010). Lateral root formation is also inhibited by the interaction of gibberellins, auxin and other plant hormones in Populus (Gou et al. 2010). Likewise, abscisic acid is also known as an inhibitor of lateral root development in Arabidopsis (De Smet et al. 2003).

Ethylene suppresses cell proliferation and root elongation through coordination with auxin signalling pathways in plants (Li et al. 2015; Street et al. 2015; Vanstraelen and Benkov 2012). qRT-PCR transcript levels of ethylene-related genes ERT and ERF were significantly increased in 12-day-old MsMIR156OE alfalfa stem cuttings, but reduced in 3-week-old and 1-month-old roots (NGS data), as the roots started to elongate at this stage. Increased qRT-PCR transcript levels for the jasmonate biosynthesis AOC gene, and the cytokinin-related COD gene were also noted in 12-day-old MsMIR156OE alfalfa stem cuttings, an increase that correlated with the raised number of main roots regenerated from stem cutting nodes of A11a and A17 genotypes. Moreover, the transcript level of COD was significantly decreased as the alfalfa roots became older suggesting that this gene operates only at the early stages of root regeneration. Promoters of both AOC3 and AOC4 jasmonate-biosynthesis genes were active in Arabidopsis roots (Stenzel et al. 2012), and elongation of roots and hypocotyls was inhibited by both cytokinin and ethylene in Arabidopsis seedlings (Cary et al. 1995). However, the mechanism underlying crosstalk between these hormones and root development is still unclear in alfalfa. Since differentially expressed genes were associated with at least six classes of phytohormones in alfalfa roots overexpressing MsmiR156, it would behoove us to elucidate the interactions of all these classes of phytohormones to find important factors that regulate alfalfa root development.

Conclusion

This study provides additional evidence as to the role that miR156 plays in regulating various aspects of growth and development in alfalfa. We previously showed that miR156 overexpression enhanced vegetative yield, delayed flowering and improved forage quality (Aung et al. 2015b). The present study revealed that miR156 may also regulate root development and nitrogen fixation activity. These findings, in addition to research reports showing that miR156 may also improve stress tolerance in plants (Cui et al. 2014) suggest that miR156 could become a valuable tool in the biotechnological improvement of alfalfa, and potentially other crops. The real value of this regulatory system may become clearer when the miR156-silenced SPL genes and other downstream genes (regulated by SPL) are characterized and linked to specific alfalfa traits. These genes would then be ideal candidates for alfalfa improvement through modern genome editing technologies, such as CRISPR-CAS9 and TILLING.

Availability of Supporting Data

All the raw sequencing data used in this study are available through the NCBI Sequence Read Archive (Accession Number: SRR2153020). The samples were sequenced as 101 bp paired-end reads on an Illumina HiSeq 2500 sequencer.