A Novel Sucrose-Regulatory MADS-Box Transcription Factor GmNMHC5 Promotes Root Development and Nodulation in Soybean (Glycine max [L.] Merr.)

Abstract

The MADS-box protein family includes many transcription factors that have a conserved DNA-binding MADS-box domain. The proteins in this family were originally recognized to play prominent roles in floral development. Recent findings, especially with regard to the regulatory roles of the AGL17 subfamily in root development, have greatly broadened their known functions. In this study, a gene from soybean (Glycine max [L.] Merr.), GmNMHC5, was cloned from the Zigongdongdou cultivar and identified as a member of the AGL17 subfamily. Real-time fluorescence quantitative PCR analysis showed that GmNMHC5 was expressed at much higher levels in roots and nodules than in other organs. The activation of expression was first examined in leaves and roots, followed by shoot apexes. GmNMHC5 expression levels rose sharply when the plants were treated under short-day conditions (SD) and started to pod, whereas low levels were maintained in non-podding plants under long-day conditions (LD). Furthermore, overexpression of GmNMHC5 in transgenic soybean significantly promoted lateral root development and nodule building. Moreover, GmNMHC5 is upregulated by exogenous sucrose. These results indicate that GmNMHC5 can sense the sucrose signal and plays significant roles in lateral root development and nodule building.

Keywords: Glycine max; GmNMHC5; MADS-box protein; lateral roots development; nodule building; sucrose.

Figure 1

Sequence alignment between GmNMHC5 and other related MADS-box proteins (Gm, G. max; At, Arabidopsis thaliana; Ms, Medicago sativa); the MADS-box and K-box domains are indicated by red and blue lines, respectively. Accession numbers are as flollows: GmNMHC5 (NP_001241489.1), MsNMHC5 (AAB51377.1), GmNMH7 (NP_001236857.1), MsNMH7 (AEW43601.1), AtAGL17 (NP_179848.1), AtANR1 (CAB09793.1).

Figure 2

Phylogenetic tree based on protein sequences between GmNMHC5 and some other function-known MIKC-type MADS-box transcription factors of Glycine max and Arabidopsis thaliana (Gm, G. max; At, Arabidopsis thaliana). Accession numbers are as follows: AtAGL17 (NP_179848.1), AtAGL21 (NP_195507.1), AtAGL16 (NP_191282.2), AtANR1 (CAB09793.1), GmNMHC5 (NP_001241489.1), GmSVP (ACJ61500.1), AtAGL15 (NP_196883.1), GmAGL15 (NP_001237033.1), GmNMH7 (NP_001236857.1), GmAGL11 (NP_001236130.1), GmAGL1 (NP_191437.1), GmAGL5 (NP_565986.1), GmAGL27 (NP_177833.3), AtAGL31 (NP 001119498.1), AtFLC (NP_196576.1), AtAGL6 (NP_182089.1), GmSEP1 (NP_001238296.1), GmAGL9 (ACA24481.1), GmMADS28 (NP 001236390.1). The Phylogenetic tree was constructed using the Maximum Likelihood method of phylogenetic tree construction, with 200 bootstrap replicates, using MEGA v.5.05. The number for each node is the bootstrap percentages, and nodes with less than 70% bootstrap values were collapsed.

Figure 3

Cellular localization of eGFP and GmNMHC5-GFP fusion proteins. 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.

Figure 4

Tissue expression pattern of GmNMHC5 revealed by real-time quantitative PCR at 29 days after SD treatment (DAT). The relative expression levels are normalized to GmCYP2. The data represent the mean ± SD of three independent experiments.

Figure 5

Expression analysis of GmNMHC5 in different tissues under SD during the growth period. The relative expression levels are normalized to GmCYP2. The data represent the mean ± SD of three independent experiments.

Figure 6

Expression analysis of GmNMHC5 in roots under different photoperiod treatment. Real-time quantitative PCR analysis of GmNMHC5 in roots at all growth periods under SD and LD, respectively. The relative expression levels are normalized to GmCYP2. The data represent the mean ± SD of three independent experiments.

Figure 7

Expression of GmNMHC5 in the root of Zigongdongdou under sucrose treatment at different stage. The X-axis represents the nine days after SD treatment plants (9 DAT), the 13 days after SD treatment plants (13 DAT) and the 29 days after SD treatment plants (29 DAT).The relative expression levels are normalized to GmCYP2. The data represent the mean ± SD of three independent experiments.

Figure 8

Overexpression of GmNMHC5 promoted the growth of lateral roots. (A) Hairy roots of the plants transformed by pGUS-GmNMHC5 or pGFPGUSPlus (as a control) via A. rhizogenes K599 with the binary vector, followed by 10 days of cultivation on 1/2 MS medium. After 5 days or 10 days of cultivation, the number of lateral roots from 30 transgenic hairy roots (B), the length of each longest lateral root from 30 transgenic hairy roots (C) and the elongation of 30 transgenic primary roots were recorded (D). The data represents the mean ± SD of three independent experiments. Means that are significantly different at the 1% (**) or 5% (*) confidence level, as detected by Student’s t-tests, are also shown.

Figure 9

Gus staining of hairy roots transformed by pGUS-GmNMHC5 and pGFPGUSPlus via A. rhizogenes K599 with the binary vector, (A) 35S::GMNMHC5-transformed hairy roots; (B) 35S::pGFPGUSPlus-transformed roots (control); (C) A segment of 35S::GMNMHC5-transformed hairy roots; (D) A segment of control roots.

Figure 10

Overexpression of GmNMHC5 promoted nodulation and nodule nitrogen fixation activity. The number of nodules from transgenic hairy roots of 30 plants and the fresh weight of 50 nodules randomly selected from the transgenic hairy roots were recorded (A); the Acetylene reduction activity (ARA) represented by the rate of ethylene production with the extension of reaction time in the transgenic nodules and the control ones were measured (B); 40 nodules randomly selected from the Gus-positive hairy roots of eight plants were used in this assay. The data represents the mean ± SD of three independent experiments. Means that are significantly different at the 5% (*) confidence level, as detected by Student’s t-tests, are also shown.