66

http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2017) 41: 66-76© TÜBİTAKdoi:10.3906/biy-1602-36

Assessment of the function and expression pattern of auxin response factor B3 in the model legume plant Medicago truncatula

Miglena REVALSKA1,*, Valya VASSILEVA2, Grigor ZEHIROV2, Sofie GOORMACHTIG3,4, Anelia IANTCHEVA1

1AgroBioInstitute, Sofia, Bulgaria2Institute of Plant Physiology and Genetics, Bulgarian Academy of Science, Sofia, Bulgaria

3Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, Ghent, Belgium4Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

* Correspondence: miglena.revalska@hotmail.com

1. IntroductionLegumes are one of the largest families of flowering crop plants with major agricultural and environmental importance, used for human food, animal feed, and production of vegetable oil (Polhill et al., 1994; Doyle and Luckow, 2003; Graham and Vance, 2003). Their ability to establish symbiotic associations with rhizobial bacteria and mycorrhizal fungi (Ane et al., 2008; Ferguson et al., 2010; Ferguson and Mathesius, 2014) and fix atmospheric nitrogen provides a substantial fraction of all nutritional proteins and reduces the need for chemical fertilizers (Cannon et al., 2006; Varshney et al., 2009). Studying the model legumes Medicago truncatula and Lotus japonicus has improved the understanding about gene function and genomic structure for legumes in general, since they are closely related to the forage species M. sativa (alfalfa) and L. corniculatus (birdsfoot trefoil) (Young and Udvardi, 2009; Iantcheva et. al., 2015).

In legume plants, the function of numerous genes regulating plant growth and development is controlled by specific transcription factor (TF) gene families, which are

strongly conserved and act as key regulators of important agronomic traits (Udvardi et al., 2007; Libault et al., 2009). TFs are involved in the control of floral meristem identity, interactions between plant roots and soil microorganisms, and responses to the environment (Ramalingam et al., 2003; Miller et al., 2006; Francia et al., 2007; Wang et al., 2013). Several TFs bind to the promoter sequences of nodulin genes, such as NIN, NSP1, and NSP2 (Schauser et al., 1999; Kalo et al., 2005; Smit et al., 2005). TFs that bind specifically to 5’-TGTCTC-3’ auxin response elements in the promoter, upstream of auxin-activated genes, are called auxin response factors (ARFs) (Ulmasov et al., 1997; Guilfoyle and Hagen, 2002, 2007; Tiwari et al., 2003). ARFs control the expression of several plant genes and contain a highly conserved plant-specific B3-type domain (Guilfoyle and Hagen, 2001; Yamasaki et al., 2004).

In Arabidopsis, 23 members of the ARF gene family participating in developmental processes have been identified (Guilfoyle and Hagen, 2007; Okushima et al., 2005). Recently, the important role of legume ARFs in governing root and nodule development has been shown

Abstract: The phytohormone auxin is a critical signal molecule, regulating fundamental processes in plant growth and development, such as shaping the root and shoot architecture, organ patterning, and nodulation. Auxin regulates plant gene expression mainly through auxin response factors (ARFs), which bind to auxin response elements in the promoter, upstream of auxin-activated genes. Here we examine and assess the function and expression pattern of a gene described as an auxin response factor, containing a DNA-binding pseudobarrel and B3 DNA-binding domains, from Medicago truncatula (МtARF-B3). For the model legume species M. truncatula, stable transgenic plants with MtARF-B3 overexpression, downregulation, and transcriptional reporters were constructed. Phenotypic and morphological evaluation of the obtained transgenic plants confirmed the important role of MtARF-B3 in general plant growth and development, modeling of root architecture, and development of seeds. Detailed histochemical and transcriptional analysis revealed expression of the gene in various stages of somatic embryogenesis, during formation of plant organs and tissues, and symbiotic nodulation. The fact that MtARF-B3 was strongly expressed in stamens and pollen grains in M. truncatula suggests that this gene could play a role in the fertility of this model legume.

Key words: Auxin response factor B3, gene expression, model legume, plant growth, plant development

Received: 09.02.2016 Accepted/Published Online: 29.05.2016 Final Version: 20.02.2017

Research Article

REVALSKA et al. / Turk J Biol

67

(Bustos-Sanmamed et al., 2013; Turner et al., 2013; Li et al., 2014; Shen et al., 2015). Based on transgenic hairy roots overexpressing miR160a and bioinformatic analysis, Bustos-Sanmamed et al. (2013) identified 30 ARFs (including one MtARF10, three MtARF16s, and 13 MtARF17s) in the M. truncatula genome. Using the latest M. truncatula reference genome sequence, Shen et al. (2015) characterized the gene structures, chromosomal locations, and expression patterns of 24 MtARFs.

This study focuses on the MtARF gene, initially identified by a reverse genetic approach in a population of Tnt1 retrotransposon-tagged mutants of M. truncatula (Revalska et al., 2011). Collaborative efforts of the Samuel Roberts Noble Foundation and partners in European groups have enabled the generation of Tnt1-insertion mutant collections for M. truncatula (d’Erfurth et al., 2003; Tadege et al, 2008; Iantcheva et al., 2009). To identify the disrupted genes in different Tnt1 mutants, flanking sequence tag information has been generated for many of the lines and deposited for public use at the Samuel Roberts Noble Foundation (http://bioinfo4.noble.org/mutant/). As confirmed by BLAST analysis (http://blast.ncbi.nlm.nih.gov/blast.cgi), the position of insertion 3 in mutant line 5928 partially corresponds to a gene encoding the auxin response factor, containing a DNA-binding pseudobarrel and B3-binding domains (Mt5g040880, subfamily ORTHO03D0200736, PLAZA 3.0 Dicots). This gene, corresponding to MtARF17d (Bustos-Sanmamed et al. 2013) or MtARF15 (Shen et al. 2015), has been named MtARF-B3. The gene was cloned, and after generation of stable transgenic plants, its expression pattern was assessed by the β-glucuronidase reporter gene (GUS). In addition, employing overexpression (OE) and RNA interference (RNAi) strategies, we analyzed the phenotypes caused by MtARF-B3 OE or downregulation, showing its crucial role in the general growth, development, and fertility of M. truncatula.

2. Materials and methods2.1. Cloning and construction of expression clones for transformationThe recombinant plasmids were obtained through GATEWAY cloning (Karimi et al., 2002) (Invitrogen Life Technologies, Inc., http://www.lifetechnologies.com). For endogenous promoter cloning, the entry clone was constructed by inserting a fragment of ~2.0 kb upstream of the start codon of the MtARF-B3 gene (Mt5g040880, PLAZA 3.0 Dicots) into the pDONRP4P1R donor vector. The expression clone was generated by recombining the entry clone into the pEX-K7SNFm14GW (promoter-NLS-GUS-GFP) destination vector possessing the neomycin phosphotransferase (nptII) gene as a selection marker for transgenic plants. To create lines overexpressing

MtARF-B3, the full-length open reading frame (ORF) of the gene was introduced into the pDONR221 donor vector. The obtained entry clone was transferred into the pK7WG2 binary destination vector for overexpression, under the control of the CaMV 35S promoter and nptII for plant selection (Karimi et al., 2007). Silencing constructs were created through the RNAi method (Limpens et al., 2003). Xwin Razor software program was used to predict in silico the region of the MtARF-B3 gene with the highest silencing capacity. The specificity of the constructs was evaluated by a single BLASTn search against the database of M. truncatula (Medicago truncatula Genome Project). The pK7GWIWG2D (II) hairpin RNA expression vector was used to create an RNAi construct using 175 bp from mRNA corresponding to nucleotide positions 1261–1431 bp of the ORF of MtARF-B3.

The expression clones were introduced into Agrobacterium tumefaciens strain C58C1, which was maintained on agar-solidified (1.5%) YEB nutrient medium supplemented with 100 mg/L rifampicin (Rif), 100 mg/L spectinomycin (Sp), and 50 mg/L gentamycin (Gm).

Primer 3 software program was used to design primers for cloning the promoter and the gene of interest (Table).2.2. Plant material, growth conditions, and genetic transformationSeeds from the highly regenerable genotype M. truncatula ‘Jemalong 2HA’ (Nolan et al., 1989) were surface-sterilized with ethanol and mercury chloride (HgCl2), rinsed with sterile distilled water (Revalska et al., 2015), and germinated on Murashige and Skoog basal medium (Murashige and Skoog, 1962). The germinated seedlings were then propagated by cuttings. In vitro plant materials were grown in Magenta boxes (60 × 60 × 96 mm, Sigma) in a growth chamber at 24 °C, with a 16-h photoperiod and light intensity of 350 µmol m–2 s–1.

Genetic transformation of the model legume was performed by a feasible Agrobacterium-mediated method of leaf disks, combining the protocols of d’Erfurth et al. (2003), Chabaud et al. (1996), and Iantcheva et al. (2009). Leaf and petiole explants collected from 35-day-old in vitro plants were used as explants. In vitro regeneration of the model legume plant M. truncatula passes through the phases of formation of callus tissue, embryo induction, development, and conversion (Revalska et al., 2015). When roots formed, plants were screened by PCR amplification for the presence of the nptII gene for Km resistance. The following gene-specific primers were used to amplify a 550-bp fragment: FW 5’-GAACAAGATGGATTGCACGC-3’ and REV 5’-GAAGAACTCGTCAAGAAGGC-3’. Positive plants were moved into a greenhouse for seed production. 2.3. Growing legume plants under hydroponic conditionsAfter germination of the transgenic and control seeds,

REVALSKA et al. / Turk J Biol

68

30-day-old seedlings were transferred to hydroponic containers containing Fahraeus nutrient solution under constant aeration and pH maintained between 6.5 and 6.8. The composition of the used solution and the terms of cultivation were reported previously (Revalska et al., 2015). The plants were inoculated with Sinorhizobium meliloti 1021 (2 × 108 cells/mL) by adding the bacterial suspension to hydroponic pots. Depending on the age of plants, the nutrient solution was changed once or twice a week.2.4. GUS activity assay Histochemical staining for GUS activity was performed essentially as described previously (Gefferson et al., 1987; Willemsen et al., 1998). Whole plants or tissues from the model species were incubated in 90% acetone for 30 min at 4 °С, then washed in phosphate buffer (pH 7.0) and incubated overnight in GUS staining solution at 37 °С. 2.5. Phenotypic analysisPhenotypic and morphological analysis of the transformed plants was performed after cultivation under in vitro, in vivo, and hydroponic conditions and compared to the wild-type (WT) M. truncatula ‘Jemalong 2HA’. Assessment of plant size (PS) and number of shoot branches (SB), root system morphology [in vitro root length (RL), number of in vitro lateral roots (LR), and root length in hydroponic conditions (HRL)], and number of nodules (N), flowers (F), and seeds (S) was conducted. Randomly selected T0 lines with the confirmed presence of the nptII gene and modified levels of MtARF-B3 transcripts were grown

under the above conditions and their progeny used for phenotype characterization and further experiments.2.6. Light microscopy Leaves, roots, and pollen samples from transgenic and WT plants were mounted on microscope slides, examined, and photographed using a Carl Zeiss Axio scope A1 microscope with differential interference contrast coupled to an XC50 digital microscope camera. 2.7. Quantitative real-time PCR (qRT-PCR) analysisTotal RNA was extracted from lines with OE and knockdown of MtARF-B3, and WT plants, using the RNeasy Plant Mini Kit (EurEx). As MtARF-B3 is a member of a multigenic family, the primers used to evaluate gene expression amplify a nonconserved region to avoid amplifying homolog genes. All primer sequences were designed with Primer 3 software program and are provided in the Table. An equal amount of total RNA was reverse-transcribed with the First Strand cDNA Synthesis Kit (Fermentas). Relative expression levels were determined with the 7300 Real-Time PCR System (Applied Biosystems). Two reference genes, actin (ACT) and ubiquitin (UBQ10), were used for data normalization. qRT-PCR analysis was performed in three biological repeats with three technical repeats each. MtARF-B3 transcript level was analyzed in T0 lines and their progeny (T1). For the RNAi lines, MtARF-B3 expression levels were determined in T0 plants. Downregulation of MtARF-B3 led to sterile plants incapable of producing T1 offspring.

Table. Primers used for cloning and qRT-PCR.

Cloning fragments

attB1 5’-GGGGACTGCTTTTTTGTACAAACTTGC-3’attB2 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-3’attB4 5’-GGGGACAACTTTGTATAGAAAAGTTGCT-3’F-ARF-B3-OE 5’-ATGTCTTCTCAGCAACGCC-3’R-ARF-B3-OE 5’-TTTTTGTGCAAGTTTGATGCC-3’F-ARF-B3-RNAi 5’-GTACAAAAAAGCAGGCTGGGAATGATAAAAGCAATGG-3’R-ARF-B3-RNAi 5’-GTACAAGAAAGCTGGGTGCTGTCAGTACCGGCAAACT-3’F-ARF-B3-pro 5’-TGTTCGAATTATATAATCGG-3’R-ARF-B3-pro 5’-GGCGGAGAGATTAACAAG-3’

qRT-PCR fragments

F-Mt-actin 5’-TCAATGTGCCTGCCATGTATGT-3’R-Mt-actin 5’-ACTCACACCGTCACCAGAATCC-3’F-Mt-Ubiquitin 5’-GCAGATAGACACGCTGGGA-3’R-Mt-Ubiquitin 5’-AACTCTTGGGCAGGCAATAA-3’F-Mt-ARF-B3 5’-CAACGGCTAAAGGTTGGTGT-3’R-Mt-ARF-B3 5’-GGACTGACTCGCTCTGGAAC-3’

REVALSKA et al. / Turk J Biol

69

2.8. Statistical analysisAll experiments were repeated three times and triplicate assays were performed for each experimental dataset. The data from qRT-PCR were analyzed by using qBase v1.3.5 software and results were expressed as mean ± standard error of the mean (SEM). Phenotypic data were expressed as mean ± standard deviation (SD), n = 4. The charts for qRT-PCR and phenotypic analysis were created by the OriginPro v8.5.1 program. Differences were assessed by t-test and considered statistically significant when P < 0.05.

3. ResultsThis study investigated the expression pattern of auxin response factor from M. truncatula (Mt5g040880, PLAZA 3.0 Dicots; MtARF-B3) in the model legume plant. According to the database PLAZA 3.0 Dicots, MtARF-B3 belongs to the gene subfamily ORTHO03D0200736, which consists of six genes in M. truncatula (Figure 1; PLAZA 3.0 Dicots Tree Explorer). The ortholog genes of Mt5g040880 are Mt2g006270 and Mt2g024430 (in chromosome 2) and Mt5g040740, Mt5g061220, and Mt5g082140 (in chromosome 5). The Mt5g040740 gene is annotated as auxin response factor 1 and the rest of the orthologs as auxin response factors. All six ortholog genes contain a DNA-binding pseudobarrel and B3-binding domains and are involved in DNA-dependent regulation of transcription and hormone responses. Their molecular function is associated with DNA-binding and protein dimerization activity. The six ortholog genes are located is the nucleus.

The function of MtARF-B3 has not been previously studied in stable transgenic plants of M. truncatula. In this study, stable transgenic T1 OE and transcriptional reporter lines and T0 RNAi lines were used. 3.1. Analysis of GUS activity in transcriptional reporter plants (pMtARF-B3::GUS-GFP) In M. truncatula, localization of MtARF-B3 expression was evaluated during the process of indirect somatic embryogenesis by GUS reporter activity. A strong GUS signal was observed in the initial explants during the process of globular embryo induction, torpedo, and subsequent early and late cotyledonary stages (Figure 2A). GUS staining was ubiquitous in the seedlings and in small in vitro plants (Figures 2B and 2C). MtARF-B3 expression was found in leaf petioles, being highest in the vascular system (Figures 2D and 2E) and in the primary root tip and lateral roots (Figures 2F–2H). A GUS signal was detected in the vascular system of nodules in plants cultivated in hydroponic conditions (Figure 2I). In the greenhouse-grown plants, GUS activity was detected in flowers, anthers and pollen, and hooks of the pods (Figures 2J–2M). 3.2. Expression profile of MtARF-B3 in M. truncatula overexpression and knockdown linesThe transcript level of MtARF-B3 was determined in several lines with MtARF-B3 OE and RNAi from M. truncatula, and part of the expression data is presented in Figure 3. The MtARF-B3 transcript level in T0 OE in vitro

Figure 1. Phylogenetic tree of the ORTHO03D0200736 subfamily in Medicago truncatula.

REVALSKA et al. / Turk J Biol

70

Figure 2. Expression pattern of pMtARF-B3::GUS in Medicago truncatula: A) different stages of somatic embryogenesis; B) seedling stage; C) in vitro plant; D) leaf; E) leaf epidermal cells and vascular system; F) in vitro root system; G) primary root primordia; H) lateral root primordia; I) nodule; J) flower petal, stamens, and stem; K) anthers; L) pollen; M) hooks of the pod.

REVALSKA et al. / Turk J Biol

71

lines of M. truncatula was significantly higher than in the WT (P < 0.001), while in T1 lines this increase reached up to 80-fold above the WT (Figure 3A). In all investigated RNAi lines, the expression level was lower than in the WT (P < 0.001) (Figure 3B). In the nodules of T1 OE lines, the MtARF-B3 expression level was higher than in the WT (P < 0.001), but it decreased in T0 RNAi lines (P < 0.001) (Figure 3C).3.3. Phenotypic and morphological analysis of M. truncatula overexpression and knockdown lines A large pool of M. truncatula T0 OE and RNAi plants from different transgenic lines was used for phenotype screening. For production of T1 seeds, some of the confirmed transgenic plants were transferred to soil under greenhouse conditions. The rest of these plants were grown hydroponically. The obtained T1 seeds from OE lines were grown on selective medium and segregated in the classical Mendelian manner. RNAi lines were sterile and therefore unable to produce offspring. After evaluation of MtARF-B3 expression levels in the T1 seedlings (Figures 3A–3C), selected T1 OE lines were used for further phenotypic analysis and production of T2 seeds. Phenotypic analysis

of four independent T1 М. truncatula ОЕ lines (Figure 4, row A) and four T0 RNAi lines (Figure 4, row B) revealed a number of growth and developmental changes compared to WT plants (Figure 4, row C). Statistical comparisons of phenotypic data are given in Figures 5A and 5B. The OE lines cultivated in vitro (Figure 4A) had a longer root length (P < 0.001) (Figure 5A) and more secondary branches along the primary root as compared to the WT plants (P < 0.001) (Figure 5B). In greenhouse conditions, the OE lines showed erect growth habit (Figure 4, row A), increased plant height (P < 0.001) (Figure 5A), and stems with a larger number of successive branches than the control 2HA (P < 0.001) (Figure 5B). In T1 OE lines, the number of formed flowers (P < 0.001) and seeds were 3 times higher than those of the WT (P < 0.001) (Figure 5B). The MtARF-B3 OE lines grown in hydroponic conditions displayed long branched roots with many effective nodules (Figure 4, row A). The root length (P < 0.05) (Figure 5A) and the number of formed nodules were significantly higher than in the WT plants (P < 0.001) (Figure 5B).

The MtARF-B3 RNAi lines grown in vitro possessed a poorly developed root system (Figure 4, row B) with

Figure 3. Relative expression level of MtARF-B3 in Medicago truncatula lines with OE, RNAi, and the WT 2HA. Three biological repeats with three technical repeats each were used. Mean expression level rates for MtARF-B3 transcript were calculated, ±SEM. A) MtARF-B3 transcript levels in T0 and T1 OE lines and WT plants; B) MtARF-B3 transcript level in T0 RNAi lines and WT plants; C) MtARF-B3 transcript level in nodules from T1 OE and T0 RNAi lines and WT plants.

REVALSKA et al. / Turk J Biol

72

shorter roots (P < 0.001) (Figure 5A) and very few branches (P < 0.05) compared to the WT (Figure 5B). Greenhouse-grown plants had a normal height, curved leaves, and small flowers (Figure 4, row B), and no significant difference

from the WT plants was found for plant height (P > 0.05) (Figure 5A) or stem branching (P > 0.05) (Figure 5B). Lines, grown in hydroponic conditions, displayed shorter, less developed, and branched root systems (Figure 4, row

Figure 4. Morphological evaluation of MtARF-B3 OE and RNAi lines of Medicago truncatula compared to the WT 2HA control plants: in vitro roots, greenhouse plant, roots of hydroponically cultivated plant, roots with nodules, flower. Row A illustrates the OE lines; row B, RNAi lines; row C, WT plants.

REVALSKA et al. / Turk J Biol

73

B) that formed fewer nodules (P < 0.001) than WT plants (Figure 5B). The flowers had irregular shapes (Figure 4, row B) and reduced number compared to the WT (P < 0.001) (Figure 5B). These abnormal flowers fell out of the calyx without forming pods and could not produce any seeds.

4. DiscussionTo analyze the quantitative effects of MtARF-B3 from the model plant M. truncatula, the gene was overexpressed in M. truncatula plants using the 35S CaMV promoter and downregulated by RNAi-mediated gene silencing technology. Additionally, nuclear-localized transcriptional reporters (pMtARF-B3::GUS-GFP) were generated, and the expression pattern of MtARF-B3 was examined. In the OE lines, the relative transcript level of MtARF-B3 was

increased, and it was downregulated in the RNAi lines, as compared to the WT. These lines allowed assessment of the MtARF-B3 function in M. truncatula and confirmed its essential role in plant growth and development.

Expression of the GUS reporter gene driven by the endogenous MtARF-B3 promoter was found at all stages of somatic embryogenesis, suggesting an involvement of MtARF-B3 in somatic embryo development. Previous research in Arabidopsis has shown that AtARF17, the closest ortholog of MtARF-B3, together with AtARF10 and AtARF16, plays an important role in controlling embryogenic development (Liu et al., 2010). The altered expression of these ARFs during embryogenesis is associated with abnormal cell divisions in the basal embryo domain and suspensor, which lead to various embryonic defects.

Figure 5. Statistical comparisons of phenotypic data for MtARF-B3 OE and RNAi lines of Medicago truncatula compared to the WT 2HA control plants. Results are expressed as mean ± SD, n = 4. A) Plant size (PS), in vitro root length (RL), and root length in hydroponic conditions (HRL); B) number of shoot branches (SB), flowers (F), seeds (S), in vitro lateral roots (LR), and nodules (N).

REVALSKA et al. / Turk J Biol

74

During postembryonic development, GUS reporter activity was detected in the vasculature of cotyledons, leaves, and stems of M. truncatula. There is substantial evidence for the role of ARFs in the differentiation of vascular tissues (Li et al., 2006). Mutation in the MONOPTEROS (MP) gene encoding the transcription factor ARF5 leads to a highly reduced leaf vein system and misaligned tracheary elements in Arabidopsis inflorescence stems and leaves (Przemeck et al., 1996).

Expression analysis using the MtARF-B3 promoter-GUS line showed a strong GUS signal in the root vascular system, developing lateral root primordia, and in the vasculature of mature nodules. These observations suggest that MtARF-B3 might be involved in both lateral root development and nodulation. It has been established that both processes have similar signaling pathways and developmental programs (De Billy et al., 2001). Legume nodulation and lateral root organogenesis initiate through divisions in the pericycle preferentially in a region near the xylem poles, and both developmental programs are tightly controlled by plant hormones, in particular by the master regulator auxin. Hence, it is not surprising that the investigated MtARF-B3 gene could be involved in both nodulation and lateral root development. The expression pattern of most MtARF genes changes significantly during S. meliloti infection, suggesting that MtARFs may activate or suppress many downstream genes involved in the nodulation process (Shen et al., 2015).

The transgenic lines with RNAi-mediated MtARF-B3 downregulation showed dramatic changes in root architecture, manifested by a reduced root length, decreased root branching, and fewer nodules as compared to the WT plants. The essential role of ARFs in the development of roots and nodules in legumes has been a subject of increasing interest. Overexpression of miR160 negatively regulates MtARF10, MtARF16, and MtARF17 transcription factors, which leads to a decreased root growth and nodule numbers in M. truncatula (Bustos-Sanmamed et al., 2013). Turner et al. (2013) overexpressed miR160 to silence these repressor ARFs in soybean and produced plants with roots hypersensitive to auxin. These roots respond to rhizobial inoculation; however, nodule primordium development is significantly reduced compared to the control. Opposite root effects were observed during MtARF-B3 overexpression. The M. truncatula T1 OE lines grown under in vitro and

hydroponic conditions formed a highly branched root system with a greater nodule number than the WT plants. Therefore, the altered root morphology and nodulation in the lines with overexpression and downregulation of MtARF-B3 could be associated with the regulatory loop miR160/ARFs, governing root and nodule organogenesis in legumes.

It should be noted that all knockdown lines produced mainly abortive flowers and had no seeds. Analysis of GUS reporter activity in M. truncatula revealed that MtARF-B3 was expressed in flowers and pollen grains. Similarly, the expression of the A. thaliana ortholog gene AtARF-B3 is observed in flowers, particularly in pollen grains (Liu et al., 2010). AtARF-B3 is essential for pollen wall formation and pollen tube growth, and it also affects plant development and fertility (Mallory et al., 2005; Liu et al., 2007; Yang et al., 2013). These findings suggest the importance of the studied MtARF-B3 gene for M. truncatula fertility, which could explain the sterility of the generated transformants with downregulation of MtARF-B3. The increased seed production in M. truncatula T1 plants with overexpression of MtARF-B3 is consistent with this assumption. Therefore, the abundance of MtARF-B3 transcripts is critical for seed production in M. truncatula.

In this study, the constructed stable transgenic M. truncatula lines with MtARF-B3 overexpression, downregulation, and transcriptional reporters allowed performing phenotypic characterization and assessment of the MtARF-B3 expression patterns. It could be concluded that MtARF-B3 was expressed at various stages of somatic embryogenesis, during formation of plant organs and tissues, in stamens, pollen, and symbiotic nodules, implying an important role of MtARF-B3 in general plant growth and development, modeling of root architecture, and development of seeds. The RNAi-mediated downregulation of this gene led to flower abnormalities and sterile plants.

AcknowledgmentsThis study was supported by a grant from the National Science Fund at the Ministry of Education and Science of the Republic of Bulgaria, project Do 02-268 “Integrated functional and comparative genomics studies on the model legumes Medicago truncatula and Lotus japonicus”. The authors are grateful to Keti Krastanova and Bistra Yuperlieva-Mateeva for valuable technical assistance.

References

Ane JM, Zhu H, Frugoli J (2008). Recent advances in Medicago truncatula genomics. Int J Plant Genomics 2008: 256597.

Bustos-Sanmamed P, Mao G, Deng Y, Elouet M, Khan GA, Bazin J, Turner M, Subramanian S, Yu O, Crespi M et al. (2013). Overexpression of miR160 affects root growth and nitrogen-fixing nodule number in Medicago truncatula. Funct Plant Biol 40: 1208-1220.

REVALSKA et al. / Turk J Biol

75

Cannon SB, Sterck L, Rombauts S, Shusei S, Cheung F, Gouzy J, Wang X, Mudge J, Vasdewani J, Schiex T et al. (2006). Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes. P Natl Acad Sci USA 103: 14959-14964.

Chabaud M, Larsonneau C, Marmouget C, Huguet T (1996). Transformation of barrel medics Medicago truncatula Gaetrn. by Agrobacterium tumefaciens and regeneration via somatic embryogenesis of transgenic plants with the MtENOD12 nodulin promoter fused to gus reporter gene. Plant Cell Rep 15: 305-310.

De Billy F, Grosjean C, May S, Bennett M, Cullimore JV (2001). Expression studies on AUX1-like genes in Medicago truncatula suggest that auxin is required at two steps in early nodule development. Mol Plant Microbe In 14: 267-277.

d’Erfurth I, Cosson V, Eschstruth A, Lucas H, Kondorosi A, Ratet P (2003). Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula. Plant J 34: 95-106.

Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM (2010). Molecular analysis of legume nodule development and autoregulation. J Integr Plant Biol 52: 61-76.

Ferguson BJ, Mathesius U (2014). Phytohormone regulation of legume-rhizobia interactions. J Chem Ecol 40: 770-790.

Francia E, Barabaschi D, Tondelli A, Laido G, Rizza F, Stanca AM, Busconi M, Fogher C, Stockinger EJ, Pecchioni N (2007). Fine mapping of an HvCBF gene cluster at the frost resistance locus Fr-H2 in barley. Theor Appl Genet 115: 1083-1091.

Guilfoyle TJ, Hagen G (2001). Auxin response factors. J Plant Growth Regul 10: 281-291.

Guilfoyle TJ, Hagen G (2002). Auxin-responsive gene expression: genes, promoters and regulatory factors. Plant Mol Biol 49: 373-385.

Guilfoyle TJ, Hagen G (2007). Auxin response factors. Curr Opin Plant Biol 10: 453-460.

Iantcheva A, Boycheva I, Revalska M (2015). Development of root tips synchronized system for the model legume  Medicago truncatula upon replication stress. Bulg J Agric Sci 21: 1177-1184.

Iantcheva A, Chabaud M, Cosson V, Barascud M, Schutz B, Primard-Brisset C, Durand P, Barker DG, Vlahova M, Ratet P (2009). Osmotic shock improves Tnt1 transposition frequency in Medicago truncatula cv Jemalong during in vitro regeneration. Plant Cell Rep 28: 1563-1572.

Jefferson RA, Kavanagh TA, Bevan MW (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907.

Kalo P, Gleason C, Edwards A, Marsh J, Mitra RM, Hirsch S, Jakab J, Sims S, Long SR, Rogers J et al. (2005). Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science 308: 1786-1789.

Karimi M, Bleys A, Vanderhaeghen R, Hilson P (2007). Building blocks for plant gene assembly. Plant Physiol 145: 1183-1191.

Karimi M, Inze D, Depicker A (2002). GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-195.

Li H, Johnson P, Stepanova A, Alonso JM, Ecker JR (2004). Convergence of signaling pathways in the control of differential cell growth in Arabidopsis. Dev Cell 7: 193-204.

Li J, Dai X, Zhao Y (2006). A role for auxin response factor 19 in auxin and ethylene signaling in Arabidopsis. Plant Physiol 140: 899-908.

Li X, Lei M, Yan Z, Wang Q, Chen A, Sun J, Luo D, Wang Y (2014). The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus. New Phytol 201: 531-544.

Libault M, Joshi T, Benedito VA, Xu D, Udvardi MK, Stacey G (2009). Legume transcription factor genes: what makes legumes so special? Plant Physiol 151: 991-1001.

Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R (2003). LysM domain receptor kinases regulating rhizobial Nod factor-induced infection. Science 302: 630-633.

Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington J (2007). Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J 52: 133-146.

Liu X, Huang J, Wang Y, Khanna K, Xie Z, Owen HA, Zhao D (2010). The role of floral organs in carpels, an Arabidopsis loss-of-function mutation in MicroRNA160a, in organogenesis and the mechanism regulating its expression. Plant J 62: 416-428.

Mallory AC, Bartel DP, Bartel B (2005). MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17: 1360-1375.

Miller AK, Galiba G, Dubcovsky J (2006). A cluster of 11 CBF transcription factors is located at the frost tolerance locus Fr-Am2 in Triticum monococcum. Mol Genet Genomics 275: 193-203.

Murashige T, Skoog F (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15: 473-497.

Nolan KE, Rose RJ, Gorst JR (1989). Regeneration of Medicago truncatula  from tissue culture: Increased somatic embryogenesis using explants from regenerated plants. Plant Cell Rep 8: 278-281.

Okushima Y, Overvoorde PJ, Arima K, Alonso JM, Chan A, Chang C, Ecker JR, Hughes B, Lui A, Nguyen D et al. (2005). Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19. Plant Cell 17: 444-463.

Przemeck GK, Mattsson J, Hardtke CS, Sung ZR, Berleth T (1996). Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200: 229-237.

REVALSKA et al. / Turk J Biol

76

Ramalingam J, Vera Cruz CM, Kukreja K, Chittoor JM, Wu JL, Lee SW, Baraoidan M, George ML, Cohen MB, Hulbert SH et al. (2003). Candidate defense genes from rice, barley, and maize and their association with qualitative and quantitative resistance in rice. Mol Plant Microbe In 16: 14-24.

Revalska M, Vassileva V, Goormachtig S, Van Hautegem T, Ratet P, Ianctheva A (2011). Recent progress in development of a Tnt1 functional genomics platform for the model legumes Medicago truncatula and Lotus japonicus in Bulgaria. Curr Genomics 12: 147-152.

Revalska M, Zehirov G, Vassileva V, Iantcheva A (2015). Is the auxin influx carrier LAX3 essential for plant growth and development in the model plants Medicago truncatula, Lotus japonicus and Arabidopsis thaliana? Biotechnol Biotec Eq 29: 786-797.

Schauser L, Roussis A, Stiller J, Stougaard J (1999). A plant regulator controlling development of symbiotic root nodules. Nature 402: 191-195.

Shen C, Yue R, Sun T, Zhang L, Xu L, Tie S, Wang H, Yang Y (2015). Genome wide identification and expression analysis of auxin response factor gene family in Medicago truncatula. Front Plant Sci 6: 73.

Smit P, Raedts J, Portyanko V, Debelle F, Cough C, Bisseling T, Geurts R (2005). NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription. Science 308: 1789-1791.

Tadege M, Wen J, He J, Tu H, Kwak Y, Eschstruth A, Cayrel A, Endre G, Zhao PX, Chabaud M et al. (2008). Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant J 54: 335-347.

Tiwari SB, Hagen G, Guilfoyle TJ (2003). The roles of auxin response factor domains in auxin-responsive transcription. Plant Cell 15: 533-543.

Turner M, Nizampatnam NR, Baron M, Coppin S, Damodaran S, Adhikari S, Arunachalam SP, Yu O, Subramanian S (2013). Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean. Plant Physiol 162: 2042-2055.

Udvardi MK, Kakar K, Wandrey M, Montanari O, Murray J, Andriankaja A, Zhang JY, Benedito V, Hofer JMI, Chueng F et al. (2007). Legume transcription factors: Global regulators of plant development and response to the environment. Plant Physiol 144: 538-549.

Ulmasov T, Hagen G, Guilfoyle TJ (1997). ARF1, a transcription factor that binds to auxin response elements. Science 276: 1865-1868.

Varshney RK, Close TJ, Singh NK, Hoisington DA, Cook DR (2009). Orphan legume crops enter the genomics era! Curr Opin Plant Biol 12: 202-210.

Wang C, Zhu H, Jin L, Chen T, Wang L, Kang H, Hohg Z, Zhang Z (2013). Splice variants of the SIP1 transcripts play a role in nodule organogenesis in Lotus japonicus. Plant Mol Biol 82: 97-111.

Willemsen V, Wolkenfelt H, de Vrieze G, Weisbeek P, Scheres B (1998). The HOBBIT gene is required for formation of the root meristem in the Arabidopsis embryo. Development 125: 521-531.

Yamasaki K, Kigawa T, Inoue M, Tateno M, Yamasaki T, Yabuki T, Aoki M, Seki E, Matsuda T, Torno Y et al. (2004). Solution structure of the B3 DNA binding domain of the Arabidopsis cold-responsive transcription factor RAV1. Plant Cell 12: 3448-3459.

Yang J, Tian L, Sun MX, Huang XY, Zhu J, Guan YF, Jia QS, Yang ZN (2013). AUXIN RESPONSE FACTOR17 is essential for pollen wall pattern formation in Arabidopsis. Plant Physiol 162: 720-731.

Young ND, Udvardi M (2009). Translating Medicago truncatula genomics to crop legumes. Curr Opin Plant Biol 12: 193-201.