The association of homeobox gene expression with stem cell formation and morphogenesis in cultured Medicago truncatula

Abstract

Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24-48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are "hijacked" for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed.

Fig. 1

Alignment of the WOX homeodomain protein sequences. Fifty-one peptide sequences from dicotyledonous species were used. AtArabidopsis thaliana, MtMedicago truncatula MtWOX1(AC137078), MtWOX3(AC169182), MtWOX4(AC148486), MtWOX5(CU326389), MtWUS(CT009654/FJ477681), MtWOX9(AC199760), Mt64(AC141864), Mt80(TC104580), Mt47(BG581947), Mt96(AC232696), Mt72(AC157472), Mt05(AC198005), Petunia (Ph) (2-EF187281, 6-PhWUS), Populus trichocarpa (P. tr)(4-AM234761, 23-AM234764, 22-AM234762, 11-AM234756, 12-AM234757, 14-AM234759, 17-AM234766, 18-AM234765), Vitis Vinifera (V. vi) (13-AM439847, 10-AM463144, 9-AM429035, 8-AM488389, 15-AM447494, 19-CAAP02003786, 1-AM488026, 21-AM463736, 20-AM486367, 24-AM435207), 3-Solanum lycopersicum (S. lyc) (FJ190667), 7-tomato(Le) LeWUS), 16-Glycine max (Gm) (DQ336954), 5-Citrus sinensis (Cs) (EU032533). Numbers at the beginning of the gene accession refer to the corresponding genes for the phylogenetic tree in Fig. 2

Fig. 2

Phylogenetic tree of WOX genes. Dendrogram based on the sequence of the homeodomains. Bootstrap (1,000 rounds) was applied and the tree drawn using Dendroscope (Huson et al. 2007) with “majority” settings for consensus. Numbers and names are the same as on Fig. 1

Fig. 3

Expression profiling by qRT-PCR of MtWUS (a) and MtWOX5 (b) in different M. truncatula tissues by qRT-PCR. The expression was investigated in the shoot apices, developing flowers (buds) and mature leaves of normally-grown plants, tips of cultured roots and somatic embryos (latter one in 2HA). Expression was related to the expression level of somatic embryos. Values are ± SE (n = 3)

Fig. 4

MtWUS RNA in-situ hybridisation in heart stage zygotic embryos (a, b), apical meristem (c) and ovules (d) at early stage of development of wild-type Jemalong. MtWOX5 RNA in-situ hybridisation in the root meristem of a seedling root (e). VC vascular cylinder (red arrows), RC root cap and black arrow indicates the quiescent centre. Bar 100 μm

Fig. 5

Expression profiling by qRT-PCR of MtWUS (a) and MtWOX5 (b) in tissue culture with different hormones. The expression was investigated in auxin plus cytokinin (Aux + Cyt, square-line), auxin alone (Aux, diamond-dash line), and cytokinin (Cyt, triangle-dot line) treatments in 35 days in 2HA. Expression calibrated to the expression level of 0 day of 2HA. Values are ± SE (n = 3)

Fig. 6

Phylogram for CLEs based on the CLE domain sequence. Fifty-five genes were analysed and are detailed in Oelkers et al. (2008) [except AC151522]. Black arrow indicates the location of MtCLV3

Fig.7

Expression profiling by qRT-PCR of MtCLV3 in different tissues of 2HA and Jemalong. Jemalong (grey bricks) and 2HA (white). Expression normalised to the expression level in somatic embryos. Values are ± SE (n = 3)

Fig. 8

Expression of MtWUS and MtCLV3 in tissue culture of Jemalong and 2HA lines with auxin plus cytokinin in the medium. The expressions of MtWUS in 2HA (square-line) and MtCLV3 in 2HA (diamond-dash line) and Jemalong (MtCLV3-Jem, triangle-dot line) were investigated over 77 days. MtCLV3 expression only occurs in the highly embryogenic 2HA line as wild-type Jemalong does not produce somatic embryos. Expression normalised to the expression level of 0 day of 2HA. Values are ± SE (n = 3)

Fig. 9

The effect of dexamethasone-induced RNAi expression for MtWUS in tissue cultures (as well as an empty vector control) developed in auxin plus cytokinin medium. The callus sizes were investigated by callus imaging and are normalised with 0 day as 100. Values are ± SE (n = 3)

Fig. 10

Transgenic calli transformed with dexamethasone-induced RNAi for MtWUS (a) and empty vector control (b) developed in auxin plus cytokinin culture. Somatic embryos can be seen in the control, but not in MtWUS RNAi transformed callus

Fig. 11

MtWUS::GUS expression at the early stages of somatic embryo induction in tissue culture. Blue-green colouring indicates the GUS signal. The signals were investigated in 3 days (a), and 14 days (b) cultured explants, 28 days callus (c), and older callus (d) with somatic embryos (white arrow). Bar 500 μm

Fig. 12

MtWUS RNA in-situ hybridisation in embryogenic callus of 2HA. The signals were investigated in whole globular stage somatic embryos. Anti-sense probe indicating the MtWUS signals (a, c). Sense probe controls (b, d). a and b are 40 μm vibratome sections while c and d are 8 μm paraffin-embedded sections. The somatic embryo (c, d) has a suspensor like structure. Bar 80 μm

Fig. 13

MtWOX5 RNA in-situ hybridisation during auxin-induced de novo root formation. The anti-sense probe (a–c). Sense probe for controls (b, d, f). The arrow labelled “1” shows centres of expression in what we have called “vein-derived” cells that emanate from the procambial cells (Rose et al. 2006). The arrow labelled “2” is pointing to the root primordium. The arrow “3” indicates the signal in the root meristem and the arrow “4” in the vascular tissue. Bar 80 μm

Fig. 14

MtWOX5::GUS expression in roots induced on auxin medium. The GUS signals in the root tip (a) and root maturation zone (b) in 8 μm paraffin-embedded sections. Two strong areas of GUS signal are indicated by red (distal) and black (proximal) arrows in a. In b, the GUS signal is indicated by a red arrow. Bar 80 μm