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. 2020 Sep 29;21(19):7180.
doi: 10.3390/ijms21197180.

Systematic Analysis of Gibberellin Pathway Components in Medicago truncatula Reveals the Potential Application of Gibberellin in Biomass Improvement

Hongfeng Wang  1   2 , Hongjiao Jiang  2 , Yiteng Xu  2 , Yan Wang  2 , Lin Zhu  2 , Xiaolin Yu  2 , Fanjiang Kong  1 , Chuanen Zhou  2 , Lu Han  2
Affiliations

Systematic Analysis of Gibberellin Pathway Components in Medicago truncatula Reveals the Potential Application of Gibberellin in Biomass Improvement

Hongfeng Wang et al. Int J Mol Sci. .

Abstract

Gibberellins (GAs), a class of phytohormones, act as an essential natural regulator of plant growth and development. Many studies have shown that GA is related to rhizobial infection and nodule organogenesis in legume species. However, thus far, GA metabolism and signaling components are largely unknown in the model legume Medicago truncatula. In this study, a genome-wide analysis of GA metabolism and signaling genes was carried out. In total 29 components, including 8 MtGA20ox genes, 2 MtGA3ox genes, 13 MtGA2ox genes, 3 MtGID1 genes, and 3 MtDELLA genes were identified in M. truncatula genome. Expression profiles revealed that most members of MtGAox, MtGID1, and MtDELLA showed tissue-specific expression patterns. In addition, the GA biosynthesis and deactivation genes displayed a feedback regulation on GA treatment, respectively. Yeast two-hybrid assays showed that all the three MtGID1s interacted with MtDELLA1 and MtDELLA2, suggesting that the MtGID1s are functional GA receptors. More importantly, M. truncatula exhibited increased plant height and biomass by ectopic expression of the MtGA20ox1, suggesting that enhanced GA response has the potential for forage improvement.

Keywords: Medicago truncatula; expression analysis; forage improvement; gibberellins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Phylogenetic analysis of GA20oxs, GA3oxs, and GA2oxs in A. thaliana (At) and M.truncatula (Mt). The proteins are grouped into four distinct groups. The phylogenetic tree was constructed using 39 protein sequences from A. thaliana (16) and M.truncatula (23) on iTOLv5 (Interactive Tree of Life; https://itol.embl.de/) online website. The red dots of different sizes (Min size: 5 px; Max size: 30 px) represent the bootstrap values, ranging from 0.5 to 1. The branches covered by different color represent the four group proteins.
Figure 2
Figure 2
Motifs composition of the MtGA20ox, MtGA3ox, and MtGA2ox proteins. The conserved motifs are predicted by MEME. The different motifs are represented by colored boxes. Motif 3 is representative DIOX_N domain. Motif 1 and motif 2 are representative 2OG-FeII_Oxy domain.
Figure 3
Figure 3
The expression patterns of MtGA20ox, MtGA3ox, and MtGA2ox genes in M. truncatula. (A) Expression analysis of gibberellin biosynthesis genes MtGA20ox and MtGA3ox in different tissues. (B) Expression analysis of gibberellin deactivation genes MtGA2ox in different tissues. R, roots; S, stems; L, leaves; SB, shoot buds; F, flowers; P, pods. The level of expression was normalized to M. truncatula UBI gene. Error bars represent SD for three biological replicates.
Figure 4
Figure 4
Phylogenetic tree analysis and exon-intron structures of the DELLA and GID1 gene family. (A) Phylogenetic analysis of DELLA and GID1 proteins from A. thaliana and M. truncatula. The tree is generated with MEGA 7.0 software using the Neighbor-Joining (NJ) method. The bootstrap analysis was conducted with 1000 iterations. The values on the phylogenetic tree represent the result of bootstrap analysis conducted with 1000 iterations. The scale bar indicates that the sequence divergence is 0.05 per unit bar, which represent 5% substitutions per nucleotide position. The empty and full circles in red color represent A. thaliana and M. truncatula DELLA proteins and the empty and full circles in green color represent A. thaliana and M. truncatula GID1 receptor proteins (B) The gene structures of the MtDELLAs and MtGID1s. Exons are represented by green boxes; introns are shown as red lines.
Figure 5
Figure 5
Expression patterns of MtDELLA (A) and MtGID1 (B) genes in six different tissues. R, roots; S, stems; L, leaves; SB, shoot buds; F, flowers; P, pods. The level of expression was normalized to M. truncatula UBI gene. Error bars represent SD for three biological replicates.
Figure 6
Figure 6
Multiple Sequence alignment of DELLA, VHYNP and SIM domains in DELLA and GID1 proteins. (A) DELLA and VHYNP domain sequences alignment for DELLA homologues in A. thaliana and M. truncatula. Black lines indicate the DELLA (left) and VHYNP (right) domain. (B) SUMO-Interaction Motif (SIM) sequences alignment for GID1 homologues in A. thaliana and M. truncatula. The black line indicates the SIM motif (W[V/I]LI). Amino acids that are conserved throughout are shaded in different colors.
Figure 7
Figure 7
Interaction tests between MtDELLA and MtGID1 proteins in the yeast two-hybrid system. Yeast transformants are spotted onto the control medium (SD/-Leu/-Trp) and selective medium (SD/-Leu/-Trp/-His/-Ade). The initial concentration of the yeast cells spots on SD/-Trp/-Leu (panel 1) and SD/-Trp/-Leu/-His/-Ade medium (panels 2 and 5) were OD600 = 0.2. Then, the yeast cells were diluted 10 and 100 times and were plated onto selective medium (panels 3, 4, 6 and 7) containing 20 μg/mL X-α-gal with or without GA3 (10−5 M).
Figure 8
Figure 8
The transcript levels of MtGA20ox, MtGA3ox, and MtGA2ox are regulated by gibberellin 3 (GA3) and its inhibitor paclobutrazol (PAC) through a feedback mechanism. (A) The transcript levels of MtGA20ox and MtGA3ox are regulated by GA3 and PAC. (B) The transcript levels of MtGA2ox are regulated by GA3 and PAC. Leaves are treated with 50 and 100 μM GA3, and 10 μM PAC. Gene expression is normalized to the control untreated (UT) expression level. Data represent the average of three independent experiments ± SD. * p < 0.05, ** p < 0.01.
Figure 9
Figure 9
Overexpression of MtGA20OX1 promoted biomass production. (A,B) The cotyledeon and leaf phenotypes of the wild type and MtGA20OX1 transgenic lines. (C) Measurements of the blade area of compound leaves on the fifth node of 4-week-old WT and MtGA20OX1 transgenic plants (means ± SD; n = 5). (D) The plant height of MtGA20OX1 transgenic and wild type plants within 50 days (means ± SD; n = 13). (E) Morphology of wild-type (WT) and MtGA20OX1 overexpressing M. truncatula plants. (F,G) The fresh and dry weight of developing wild type and MtGA20OX1 transgenic plants (means ± SD; n = 10). Bars = 1 cm in A and B. ** p < 0.01, *** p < 0.001.
Figure 10
Figure 10
Expression analysis of cell development related genes in wild-type and MtGA20OX1 transgenic lines. (A–C) The expression of cyclindependent protein kinase genes, MtCYCB1.1, MtCYCB1.2, and MtCYCD2.1. (D–F) The expression of MtKRP1, MtKRP2, and MtKRP3 genes. ** p < 0.01, *** p < 0.001.
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