GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean (Glycine max [L.] Merr.)

Journal of Integrative Agriculture 2019, 18(3): 553–562 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean (Glycine max [L.] Merr.) MA Wen-ya1*, LIU Wei1*, HOU Wen-sheng1, SUN Shi1, JIANG Bing-jun1, HAN Tian-fu1, FENG Yong-jun2, WU Cun-xiang1 1

Key Laboratory of Soybean Biology (Beijing), Ministry of Agriculture/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 School of Life Science, Beijing Institute of Technology, Beijing 100081, P.R.China

Abstract As an important food crop and oil crop, soybean (Glycine max [L.] Merr.) is capable of nitrogen-fixing by root nodule. Previous studies showed that GmNMH7, a transcription factor of MADS-box family, is associated with nodule development, but its specific function remained unknown. In this study, we found that GmNMH7 was specifically expressed in root and nodule and the expression pattern of GmNMH7 was similar to several genes involved in early development of nodule (GmENOD40-1, GmENOD40-2, GmNFR1a, GmNFR5a, and GmNIN) after rhizobia inoculation. The earlier expression peak of GmNMH7 compared to the other genes (GmENOD40-1, GmENOD40-2, GmNFR1a, GmNFR5a, and GmNIN) indicated that the gene is related to the nod factor (NF) signaling pathway and functions at the early development of nodule. Over-expression of GmNMH7 in hairy roots significantly reduced the nodule number and the root length. In the transgenic hairy roots, overexpression of GmNMH7 significantly down-regulated the expression levels of GmENOD40-1, GmENOD40-2, and GmNFR5α. Moreover, the expression of GmNMH7 could respond to abscisic acid (ABA) and gibberellin (GA3) treatment in the root of Zigongdongdou seedlings. Over-expressing GmNMH7 gene reduced the content of ABA, and increased the content of GA3 in the positive transgenic hairy roots. Therefore, we concluded that GmNMH7 might participate in the NF signaling pathway and negatively regulate nodulation probably through regulating the content of GA3. Keyword: soybean, GmNMH7, MADS-box gene, nodulation, ABA, GA3

1. Introduction Received 28 December, 2017 Accepted 2 May, 2018 Correspondence FENG Yong-jun, Tel: +86-10-68914495-804, Fax: +86-10-68915956, E-mail: [email protected]; WU Cunxiang, Tel: +86-10-82105865, Fax: +86-10-82108784, E-mail: [email protected] * These authors contributed equally to this study. © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/) doi: 10.1016/S2095-3119(18)61992-6

Leguminous plants can utilize nitrogen in the atmosphere by nodulating. Root nodules initiate from the interaction between host plants and nitrogen-fixing rhizobia. Host plants generate flavonoids to attract rhizobia to migrate towards the root and stimulate it to secrete nod factors (NFs) (Sanjuan et al. 1992), a lipochito-oligosaccharide signal molecule, which is very essential for initiation and development of nodule. NFs will be perceived and accepted by nod factor receptors (NFRs) (Broghammer et al. 2012),

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encoded by LjNFR1 and LjNFR5 in Lotus japonicas (Madsen et al. 2003; Radutoiu et al. 2003), MtLYK3 and MtNFP in Medicago truncatula (Amor et al. 2003; Limpens et al. 2003), PsSym10 in Pisum sativum (Geurts et al. 1997; Madsen et al. 2003), and GmNFR1α, GmNFR1β, GmNFR5α, and GmNFR5β in Glycine max (Indrasumunar et al. 2010, 2011). NFRs have been proven to combine with NFs directly (Broghammer et al. 2012), and promote root hair deformation, infection threads formation, cortical and pericycle cell division, and nodule primodium. The first morphological change in root epidermis induced by rhizobia is root hair deformation or branching (Heidstra et al. 1994). Rhizobia get into cell through the deformed epidermis, and gradually deep into the cortex cells via the formed infection threads (ITs) (Velde et al. 2006). When ITs extend to nodule primodium in cortical, rhizobia will be released into nodule primodium cell, and differentiate into nitrogen-fixing bacteroid, which can form symbiosome with nodule primodium and subsequently develop into nodules. This pathway of regulating nodulation is called NF signaling pathway. ENOD40 genes are the crucial component in NF signaling pathway and were identified as the earliest nodulin genes (Yang et al. 1993; Crespi et al. 1994; Mathesius et al. 2000; Compaan et al. 2001). The expression of ENOD40 genes can significantly affect the number of nodules in legume plants, mainly through affecting the formation of nodule primodium and the development of nodules rather than the formation of ITs (Charon et al. 1999; Kumagai et al. 2006; Wan and Franssen 2007). A root nodules-specific gene, NMH7, was firstly cloned from root nodules in alfalfa. It is homologous to AP3 in Arabidopsis thaliana and DEFA gene in Antirrhinum (Páez-Valencia et al. 2008a, b). The NMH7 protein appeared before rhizobium inoculation demonstrated that NMH7 gene is involved in non symbiotic events and the colonization of rhizobia and nodule growth process (PáezValencia et al. 2008a). It is proved that alfalfa fructose1,6-bisphosphate aldolase and NMH7 protein located in cytoplasm and nucleus of epidermis cells simultaneously, and fructose-1,6-bisphosphate aldolase is a MADS domain binding protein, which is the earliest report about the direct interaction between glycolytic enzyme and transcription factors of MADS-box family (Páez-Valencia et al. 2008b). It is demonstrated that NGL9 protein’s capability of binding to DNA relies on NMH7, and they functionate by forming heterodimer (Zuccheroet al. 2001). In soybean (Glycine max [L.] Merr.), GmNMH7 (NM_001249928) is the homologous gene of NMH7 in alfalfa. It located in chromosome 6, containing seven exons and six introns, encoding a transcription factor including MADS-box and K-box domains. In situ hybridization experiments to determine in situ expression of GmNMH7

were performed in Zigongdongdou (ZGDD), a highly photoperiod-sensitive variety, and found different expression pattern, under different photoperiods. GmNMH7 expresses strongly in leaf and flower primordia, and floral organs but the expression in root nodule was repressed under short day condition, whereas the flowering process is promoted. Under long day condition, the flowering process is inhibited, and the expression of GmNMH7 is enhanced in root nodules and repressed in other tissues tested (Wu et al. 2006). These facts illustrate that GmNMH7 may be related to nodulation process, and may even play an important role in nodulation. However, the specific function of GmNMH7 in nodulation remained unclear. In this study, we measured the expression pattern of GmNMH7 and found that this gene may function at the early development of nodules. Its overexpression in hairy roots reduced the nodule number and the root length and down-regulated the expression levels of several genes involved in the early development nodules.

2. Materials and methods 2.1. Plant materials A photoperiod-sensitive variety ZGDD cultivated in 1:1 mixed soil and vermiculite, 24°C under the condition of 60% relative humidity. 8 h light/16 h dark of short day treatment and 16 h light/8 h dark of long day treatment were conducted respectively according to the growth characteristics of the variety (Wu et al. 2006).

2.2. Bacteria materials Bradyrhizobium japonicum (B. japonicum) USDA110 was used for this experiment to induce root nodules.

2.3. Cloning of GmNMH7 GmNMH7 coding sequence was cloned from the nodules of ZGDD using high fidelity enzyme KOD-plus-Neo (Toyobo, Osaka, Japan), template of ZGDD nodule cDNA library and primers designed according to GmNMH7 coding sequence provided by the NCBI database.

2.4. Analysis of gene expression Expression analysis of GmNMH7 ZGDD were treated at short day condition. Roots, root nodules, stems, leaves, flowers, and shoot apices were sliced off at 28 days after short day treatment. Total RNA was extracted from the samples using Trizol Up Kit (TransGen Biotech, Beijing, China) and then was transcribed into cDNA using TransScript II One-Step gDNA Removal and cDNA Synthesis Super Mix

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(TransGen Biotech, Beijing, China). Real-time PCR was performed on ABI7900 (Applied Biosystems, Foster City, CA, USA) using TaKaRa SYBR Premix Ex Taq Kit (TaKaRa, Kusatsu, Japan) and cDNA library as template. And the internal reference is GmACTIN. The data were analysed by SDS2.3 Software (Applied Biosystems, Foster City, CA, USA), and every sample was repeated three times. Analysis of GmNMH7 gene expression under rhizobia inoculating treatment 2-week-old ZGDD seedlings were inoculated with rhizobia under short day condition. Roots were harvested at 0, 1, 3, 6, 9, 12, 24, and 72 hours postinoculation (hpi), 3, 5, and 10 days post-inoculation (dpi), respectively. And total RNA was extracted from these samples and real-time PCR was conducted same as the section of expression analysis of GmNMH7. Analysis of GmNMH7 gene expression under phytohormone treatment Seedlings of 2-week-old ZGDD, planted under short day condition, were soaked for 3 hours in different concentrations of ABA and GA3 (0, 10, 50, and 100 μmol L–1). The samples of roots were harvested from three seedlings. And total RNA was extracted from these samples and real-time PCR was conducted same as the section of expression analysis of GmNMH7.

2.5. Bioinformatic analysis of the GmNMH7 promoter A sequence of 2 500 bp ahead to the first ATG of GmNMH7, obtained from the phytozome database (http://www. phytozome.net/) was viewed as the promoter sequence of GmNMH7 (GmNMH7pro). Bioinformatic analysis of GmNMH7pro for exploring potential function was performed using an online Software PLACE (http://www.dna.affrc.go.jp/ PLACE/signalup.html).

2.6. Construction of 35S::GmNMH7 for overexpressing the gene For over-expression of GmNMH7 in hairy roots, we combined GmNMH7 to linearized pGFPGUSPlus vector digested by SacI and XbaI enzyme using pEASY®-Uni Seamless Cloning and Assembly Kit (TransGene Biotech, Beijing, China), so that GmNMH7 can be driven by the CaMV 35S promoter.

2.7. Transformation of hairy roots and rhizobia inoculation assay For exploring the function of GmNMH7 during root and nodule development, K599 strain containing 35S::GmNMH7 was injected into 1-week-old ZGDD seedlings through stabbing at the cotyledonary node twice with a syringe needle (Cao et al. 2011). The K599 strain containing

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pGFPGUSPlus was used as a control inoculant. The main roots of the plants were removed 7 days after the hairy roots emerging. The plants with the hairy roots were transferred into new pots with wet vermiculite and watered with 200 mL of fahraeus nitrogen-free nutrient solution (Fåhraeus et al. 1957) per pot at 7 day-interval, and inoculated with 1 mL of B. japonicum USDA110 culture (OD600=0.6) after 5 days of transplanting.

2.8. Phytohormone assay To explore the influence of over-expressing GmNMH7 on the contents of ABA and GA3, we measured the contents of ABA and GA3 in the transgenic hairy roots using ELISA method (Wang et al. 1995; Patel and Thaker 2007). Fifty transgenic hairy roots were used in the measurement.

3. Results 3.1. GmNMH7 had high identity with MsNMH7 The coding sequence of GmNMH7 obtained from the NCBI database was cloned using nest PCR method. Protein encoded by GmNMH7 gene was predicated to be a 227 amino acids sequence, and shared 89.96% identity with MsNMH7 and 54.74% identity with AtAP3. All the sequences include a MADS_MEF2 (myocyte enhancer factor 2) _like and a K-box structure domain, and all belong to the second class by MADS-box protein (Fig. 1). The upstream of the GmNMH7 (2 500 bp) was analyzed by applying an online software (http://www.dna.affrc.go.jp/ PLACE/signalup.html). The prediction result showed that besides the basic elements such as TATA box and CAAT box, there were many cis-acting elements related to nodulation and hormone response in GmNMH7pro (Table 1), suggesting that GmNMH7 gene may be associated with the rhizobia infection and the development of root nodule. The subcellular localization of GmNMH7 was predicted by online Software BaCelLO (http://gpcr.biocomp.unibo.it/ bacello/). The results showed that GmNMH7 was localized in the nucleus and cytomembrane. In order to explore the expression position of GmNMH7 intracellularly, the subcellular localization of GmNMH7 was studied. We cloned GmNMH7 gene coding sequence, which is removed the terminator, and constructed plasmid with expressed GmNMH7-GFP. A transient expression assay of the fused protein (GmNMH7-GFP) was carried out in onion epidermal cells. Confocal microscopy observations suggested that the GmNMH7-GFP fusion protein was located in the cytomembrane and nucleus (Fig. 2).

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MADS_MEF2_like region GmNMH7 MsNMH7 AtAP3 K-box region

GmNMH7 MsNMH7 AtAP3 GmNMH7 MsNMH7 AtAP3 GmNMH7 MsNMH7 AtAP3

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Fig. 1 Sequence alignment among GmNMH7, MsNMH7, and AtAP3. MADS_MEF2 (myocyte enhancer factor 2)_like and K-box were indicated by black and red lines, respectively. Accession numbers are as follows: GmNMH7 (NP_001236857.1), MsNMH7 (AEW43601.1), and AtAP3(NP_191002). Table 1 Several cis-acting elements of GmNMH7 promoter Cis-element name OSE1ROOTNODULE NODCON1GM TATABOX4 CAATBOX1 GTGANTG10 POLLEN1LELAT52 GAREAT CATATGGMSAUR SURECOREATSULTR11 MYC

A

B

Signal sequence (5´→3´) AAAGAT AAAGAT TATATAA CAAT GTGA AGAAA TAACAAR CATATG GAGAC CANNTG

Function Related to the infection of rhizobia Nodulin gene Combined with RNA polymerase, start transcription Regulating transcription efficiency Promoter element of late pollen gene Pollen specific activation response element Gibberellin response element Auxin response element Sulfur response element Abscisic acid, low temperature stress response element

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Fig. 2 Cellular localization of green fluorescent protein (GFP) and GmNMH7-GFP fusion proteins. Transient expression of GFP and GmNMH7-GFP in onion epidermal cells. Photographs were taken in a dark field for green fluorescence (left column) and a bright field for cell morphology (middle column); the right column is the overlapping view of the dark and bright fields for comparative clarity.

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3.2. Expression of GmNMH7 was involved in the early stage of nodulation

To identify whether GmNMH7 is involved in early stage of symbiotic interaction or the initiation of nodulation, a real-time quantitative PCR analysis was carried out to measure the expression level of GmNMH7 after 10 days of inoculating with rhizobia. The expression of GmENOD40-1, GmENOD40-2, GmNIN, GmNFR1α, and GmNFR5α, which were related to early nodulation, was also measured at the same time. As shown in Fig. 4, the expression of GmNMH7 and those nodulation-related genes were all peaking after rhizobia inoculation. However, the peak of GmNMH7 expression appeared earlier (9 hous after inoculation) than other genes (12 or 24 hours after inoculation) (Fig. 4). It was certain that GmNMH7 can respond to the rhizobia in a more sensitive time limit, and may participate in the rhizobium infection or nodule initiation process.

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3.3. Over-expression of GmNMH7 decreased nodulation

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Considering that GmNMH7 presented a higher expression level in root and nodule and could response to rhizobia inoculation, we further determined its function in nodule development. The Agrobacterium rhizogenes K599 strains containing 35S::GmNMH7 and pGFPGUSPlus were used to induce hairy roots to obtain the over-expressing GmNMH7 and control transgenic roots, respectively. Then those transgenic roots were inoculated by rhizobia. The positive transgenic hairy roots were validated by GFP

0.1 Leaf

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Tissues of Zigongdongdou

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Fig. 3 Tissue expression pattern of GmNMH7 revealed by realtime quantitative PCR at 28 days after short day (SD) treatment. The relative expression levels are normalized to GmACTIN. The data represent the mean±SD of three independent experiments.

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The expression levels of GmNMH7 in different tissues (roots, nodules, stems, leaves, flowers, and shoot apices) were performed by real-time PCR (Fig. 3). It is revealed that the expression of GmNMH7 can be detected in all of the tissues. However, its expression in root and nodule was about 2.4, 12, 190, and 60 times more than that in stems, leaves, flowers, and shoot apices, respectively, showing that GmNMH7 may be associated with the development of root and root nodule.

D

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Fig. 4 Relative expression level of GmNMH7 (A) and genes (B, GmENOD40-1; C, GmENOD40-2; D, GmNIN; E, GmNFR1α; F, GmNFR5α) regulating nodulation in early stage in root at various times after inoculation (hours/days post-inoculation (hpi/dpi)). The relative expression levels are normalized to GmACTIN. The data represent the mean±SD of three independent experiments.

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The expression pattern of GmNMH7 induced by rhizobia implied that it was involved in NF signal pathway. We wondered if GmNMH7 regulated the activity of other components in NF signal pathway. Then, the expression levels of GmENOD40-1, GmENOD40-2, GmNIN, and GmNFR5α in the positive transgenic hairy roots were determined. GmENOD40 genes are crucial in NF signal pathway and play an important role in the nodule primordium

observation and the nodule number and main root length were investigated subsequently at 28 days after inoculation (Fig. 5). As shown in Fig. 6, the average number of nodules in positive transgenic hairy roots was much lower (17.82) than that in the control hairy roots (29.96) (Fig. 6-A, B, D). Furthermore, the length of the 35S::GmNMH7 transformed tap roots was also shortened significantly compared to that of the control (Fig. 6-C and D).

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Fig. 5 Green fluorescent protein (GFP) image of positive transgenic hairy root. A, fluorescence field. B, bright field. PX, pixel.

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Fig. 6 Over-expression of GmNMH7 decreased the nodule number. A, phenotype of over-expressing GmNMH7. B, average nodule number per cm of transgenic hairy roots. C, average length of transgenic hairy roots. D, expression level of GmNMH7 in transgenic hairy root. The relative expression levels are normalized to GmACTIN. 35S::GmNMH7, over-expressing GmNMH7; EV, empty vector. Statistical significance was determined using Student’s t-tests, SAS (*, P<0.05; **, P<0.01). The data represented the mean±SD of three independent experiments.

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initiation. GmNFR5α encodes a kind of protein called NFRs. Our results revealed the significant decreased expression of GmENOD40-1, GmENOD40-2, and GmNFR5α, indicating an opposite expression tendency between GmNMH7 and these genes (Fig. 7).

3.4. Over-expressing GmNMH7 decreased the content of ABA but increased the content of GA3 Given the fact that there are numerous ABA and GA 3 response cis-acting elements in the GmNMH7pro sequence (Table 1), one will ask whether GmNMH7 could respond to these 2 phytohormones, therefore different concentrations of exogenous ABA and GA3 were applied to treat the roots of two-week-old seedlings. Real-time PCR data showed different patterns of GmNMH7 expression under ABA and GA3 treatments. Compared to the control, ABA down-regulated the expression level of GmNMH7, yet GA 3 regulated the expression of GmNMH7 in a dual manner, with eliciting higher expression of GmNMH7 at lower concentration while inhibiting the expression at higher concentration (Fig. 8). It can be deduced that the expression pattern of GmNMH7 could response to ABA and GA3. Simultaneously, we also determined the contents of ABA and GA3 in the GmNMH7-over-expressing transgenic hairy roots and revealed that over-expressing GmNMH7 decreased the content of ABA but increased the content of

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It was proven that MADS-box genes play multiple roles in different plant development processes. In this study, we studied GmNMH7, a MADS-box gene, and found that it showed high identity with MsNMH7 and AtAP3. Results of subcellular localization showed that GmNMH7 protein located in the cell membrane and nucleus (Fig. 2). GmNMH7 showed a relative higher expression in root and nodule (Fig. 3), which excited our attention on the possible function of GmNMH7 in nodulation. Expression pattern of GmNMH7 gene within 10 days after rhizobia inoculation was shown to be similar to GmENOD40-1, GmENOD40-2, GmNIN, GmNFR1α, and GmNFR5α, several genes regulating early development of nodule. Even more, the expression peak of GmNMH7 gene was earlier than the other genes, indicating that the gene is related to the NF signaling pathway and that the gene is likely to function at the early development of nodule. NF signaling pathway plays crucial role in regulating nodule development. Consequently, the expression of GmNMH7 was induced by

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4.1. The GmNMH7 gene may inhibit the nodulation by reducing the expression of GmENOD40-1, GmENOD40-2, and GmNFR5α genes

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4. Discussion

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GA3 significantly (Fig. 9).

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Fig. 7 The expression level of GmENOD40-1 (A), GmENOD40-2 (B), GmNIN (C), and GmNFR5α (D) in transgenic roots. The relative expression levels are normalized to GmACTIN. 35S::GmNMH7, over-expressing GmNMH7; EV, empty vector. The data represent the mean±SD of three independent experiments. Statistical significance was determined using Student’s t-tests, SAS (**, P<0.01).

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B Relative expression of GmNMH7

Relative expression of GmNMH7

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Fig. 8 Relative expression level of GmNMH7 under abscisic acid (ABA, A) and gibberellin (GA3, B) treatments. The relative expression levels are normalized to GmACTIN. The data represent the mean±SD of three independent experiments.

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Fig. 9 Content determination of abscisic acid (ABA, A) and gibberellin (GA3, B) in transgenic hairy roots. 35S::GmNMH7, overexpressing GmNMH7; EV, empty vector. The data represent the mean±SD of three independent experiments. Statistical significance was determined using Student’s t-tests, SAS (**, P<0.01).

rhizobia, and we speculated that GmNMH7 is a component of NF regulation pathway. To explore the specific function of GmNMH7 during nodulation, we over-expressed GmNMH7 in the hairy roots, and observed obviously reduced number of nodule and notably down-regulated expression levels of GmENOD40-1, GmENOD40-2, and GmNFR5α. It is speculated that the GmNMH7 gene may suppress the nodulation by reducing the expression of GmENOD40-1, GmENOD40-2, and GmNFR5α genes. But the mechanism of the regulation remains unknown. In our further study, the genome-editing tools such as CRISPR/Cas9 and RNAi can be used to confirm its function on nodulation.

4.2. Up-regulation of GA3 might be one reason for GmNMH7 to suppress nodule number in this study Previous studies have proven that exogenously-added GA3 decreased nodule number in L. japonicas (Maekawa et al. 2009) and exogenously-added GA3 suppressed the

expression level of NSP2, a gene required for the initiation of nodule and functions upstream of GmENOD40-1 and GmENOD40-2 (Schauser et al. 1999). In the current study, over-expressing GmNMH7 reduced the number of nodule and increased the content of GA3 in the transgenic roots. It was proved that GAs regulate the nodulation negatively in soybean (Williams and Mallorca 1984; Feng et al. 1997). Thus, we concluded that GmNMH7 might inhibit nodule number by up-regulating GA3, but the specific functional mechanism is still obscure and would be an important work in future studies.

4.3. GmNMH7 could contribute to preventing too much energy-consuming resulted by too many nodules, thus to optimize the growth of whole soybean plant Nodulation is a very important process for legumes production. However, nodule formation and nitrogen fixation

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are also energy-consuming processes, and legumes have to develop the minimal number of nodules required to ensure optimal growth. This study revealed that GmNMH7 could regulate nodulation negatively, suggesting that GmNMH7 can contribute to preventing too many nodules and also the resulted too much energy-consuming, thus to improve the growth of whole soybean plant.

5. Conclusion In this study, we found a MADS-box gene, GmNMH7, could inhibit root development and nodulation of soybean. Furthermore, over-expressing GmNMH7 decreased the content of ABA but increased the content of GA3.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31271636) and the earmarked fund for China Agriculture Research System (CARS-04). We appreciate Dr. Li Jun (Chinese Academy of Agricultural Sciences) for providing B. japonicum USDA110, Prof. Peter Gresshoff (University of Queensland, Australia) for providing A. rhizogenes strain K599 and the binary vector pGFPGUSPlus.

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