Search

The common ice plant, Mesembryanthemum crystallinum L. provides a useful model for the study of environmentally induced photosynthetic conversion and abiotic stresses tolerance. However, a procedure for the production of transgenic ice plant, which is essential for functional genomics, has not been fully established. Here we tested the factors on the transformation of cotyledonary nodes excised from the ice plant seedlings such as thidiazuron (TDZ), NaCl and phytosulfokine (PSK), a peptidyl plant growth factor using Agrobacterium tumefaciens strains EHA101 and EHA105 harboring binary vector plasmids pBI7EGFP and pCAMBIA1302, respectively. The established procedure is as follows: the explants (cotyledonary nodes) were co-cultivated with Agrobacterium for 3 days, and the explants were cultured in the medium with 0.5 mg l−1 kinetin and 100 mg l−1 carbenicillin for 72 h, and they were cultured in the medium with 0.5 mg l−1 kinetin and 100 nM PSKfor 4 weeks. Thidiazuron and NaCl enhanced the production of multiple adventitious shoot formation during regeneration but reduced the transformation efficiency due to the vitrification of adventitious shoots. PSK was effective in the production of healthy adventitious shoots. The transformation frequency at the stage of whole plants was 0.6% and 4.6% per inoculated cotyledonary nodes using the Agrobacterium strain EHA101 (pBI7EGFP) and EHA105 (pCAMBIA1302), respectively.

A halophyte, the common ice plant (Mesembryanthemum crystallinum L.), shows the maximal growth under salinity, in which almost all crops die. The NaCl-stimulated growth, which is referred to as halophilism, is an important trait for adaptation to salinity, but the mechanism is still unclear. To elucidate factors contributing to the halophilism, we tested the effects of NaCl on growth, ion accumulation, and expression of ion homeostasis-related genes in suspension-cultured cells. Among nine ions analyzed, Na+, Cl−, K+, and NO3− were accumulated significantly in the cells showing halophilism than that in the salt-untreated cells, and the accumulation of these ions was positively correlated with the growth. Also, the expression of ion homeostasis-related genes for plasma membrane transporters and channels for incorporation of nitrate (McNRT1), sodium (McHKT1), potassium (McKmt1), cations/Cl− (McCCC1), for tonoplast antiporters H+/Cl− (McCLC1) and Na+/H+ (McNHX1), and V-ATPase subunit c (McVmac1) for sequestration of Cl− and Na+ into the vacuole, and for enzymes catalyzing biosynthesis of proline (McP5CS) and ononitol (McImt1) was higher in the cells showing halophilism than that in the salt-untreated cells. These results indicate that the ion accumulation and the expression of ion homeostasis-related genes contribute to the NaCl-stimulated growth enhancement in the halophyte, the common ice plant.Abbreviations: CCC: cation/Cl- cotransporter; CLC: H+/Cl–antiporter; DW: dry weight; FW: fresh weight; HAK: high affinity potassium; HKT: high potassium transporter; Imt: myo-inositol O-methyl transferase; MIP: major intrinsic protein; NHX: Sodium/proton antiporter; P5CS: delta 1-pyrroline-5-carboxylate synthase; PCV: Packed cell volume; PEG: polyethylene glycol; XTH: xyloglucan endotransglucosylase/hydrolase

The common ice plant, Mesembryanthemum crystallinum L., a halophytic new functional leafy vegetable crop, develops epidermal bladder cells (EBCs) on the surfaces of its aerial organs. Our previous studies of the physiological characteristics of the wild-type and the EBC-less mutant indicated that EBCs sequester salt and maintain ion homeostasis within photosynthetically active tissues. The EBC has been thought to be a modified trichome; however, molecular mechanisms governing EBC development in the common ice plant have not been fully understood. Here, we have analyzed the steady-state mRNA abundance of nineteen cotton fiber-related gene homologs and eight Arabidopsis trichome development-related genes, and found that a MYB transcription factor homolog (McMYB2) and a GLABRA2-like gene (McC4HDZ) were preferentially expressed in wild-type plants, whereas a putative TRIPTYCHON (McTRY)- and CAPRICE-like gene (McCPC) were preferentially expressed in the EBC-mutant. The full-length cDNA sequences of these homologs were determined, and constructs containing McC4HDZ and McMYB2 were introduced into an Arabidopsis trichome-less mutant and wild-type plants. Overexpression of McMYB2 in wild-type Arabidopsis increased trichome number, associated with activation of the trichome development-related gene, GLABRA2 (GL2). Moreover, overexpression of McC4HDZ partially complemented trichome development in the trichome-less mutant of gl2-1, and resulted in increased trichome number in wild-type Arabidopsis, associated with the upregulation of key trichome-positive regulators GLABRA1 (GL1) and TRANSPARENT TESTA GLABRA1 (TTG1). These results suggest that McMYB2 and McC4HDZ could be functional in Arabidopsis trichome formation, implying that EBCs of the common ice plant and trichomes of Arabidopsis may share some molecular mechanisms in their development.

Mesembryanthemum crystallinum L., a halophytic species, displays modified trichomes, epidermal bladder cells (EBC), on the surfaces of its aerial organs. EBCs serve to sequester excessive salt from underlying metabolically active tissues. To elucidate the molecular determinants governing EBC development in the common ice plant, we constructed a cDNA-based suppression subtractive hybridization library and identified genes differentially expressed between the wild-type and the EBC-less mutant. After hybridization, 38 clones were obtained. Among them, 24 clones had homology with plant genes of known functions, whose roles might not be directly related to EBC-morphology, while 14 clones were homologous to genes of unknown functions. After confirmation by northern blot analysis, 12 out of 14 clones of unknown functions were chosen for semi-quantitative RT-PCR analysis, and the results revealed that three clones designated as MW3, MW21, and MW31 preferentially expressed in the EBC-less mutant, whereas the other two designated as WM10 and WM28 preferentially expressed in the wild type. Among these genes, the expression of a putative jasmonate-induced gene, designated as WM28 was completely suppressed in the EBC-mutant. In addition, the deletion of C-box cis-acting element was found in the promoter region of WM28 in the EBC-less mutant. Overexpression of WM28 in Arabidopsis resulted in increased trichome number due to the upregulation of key trichome-related genes GLABRA1 (GL1), and GLABRA3 (GL3). These results demonstrate that WM28 can be an important factor responsible for EBC formation, and also suggest the similarity of developmental mechanism between trichome in Arabidopsis and EBC in common ice plant.