Suppression subtractive hybridization library construction and identification of epidermal bladder cell related genes in the common ice plant, Mesembryanthemum crystallinum L.

Abstract

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.

Making Plants Break a Sweat: the Structure, Function, and Evolution of Plant Salt Glands

“…Some of the lineage specific transcripts are easily detected in the epidermal bladder cell transcriptomes at high expression levels, but appear to be expressed at low levels or are undetected in whole shoot transcriptomes, indicating the importance of studies of individual salt gland cell types (Oh et al, 2015). Genes specific to bladder cell function and development that were identified using a suppression subtractive hybridization library construction between wild type M. crystallinum and mutant plants without bladder cells also revealed a significant number of lineage specific genes with unknown functions (Roeurn et al, 2016). One such gene of unknown function detected via the comparison between wildtype and mutant plants was subsequently overexpressed in Arabidopsis , resulting in a phenotype with an increased number of trichomes on leaves, and this gene was inferred to regulate trichome initiation via regulating GL2 in the trichome development pathway (Roeurn et al, 2016).…”

MYB and HD-ZIP IV homologs related to trichome formation are involved in epidermal bladder cell development in the halophyteMesembryanthemum crystallinumL.

“…The increased expression of McMYB2 resulted in the increased number of trichomes in the transgenic Arabidopsis (Figure 4). In Arabidopsis, it has been suggested that R2R3 MYBs (GL1, MYB5, and MYB23) interact with a bHLH protein (GL3 and that WM28 positively regulated trichome in Arabidopsis by increase of the expression of genes for trichome development-acting complex, GL1 and GL3 (Roeurn et al, 2016). In the present study, we attempt to further elucidate the molecular genetic basis of the development of EBCs in the common ice plant through the analysis of the common ice plant homologs of the trichome and fiber-development related-genes of Arabidopsis and cotton.…”

“…Development of cotton fibers takes place from 0 to 60 d post-anthesis (DPA) that including fiber initiation, elongation, biosynthesis of secondary cell walls, and maturation (Lee et al, 2007;Li et al, 2002). Genes for several transcription factors that function in fiber development, including GaMYB2, GhMYB25, GhMYB109, and a putative homeodomain transcription factor GhHD1, which are preferentially expressed in early fiber initiation, have been identified (Suo et al, Wild type of Arabidopsis thaliana (ecotype Col-0) and the gl2-1 mutant in Landsberg erecta (Koornneeff et al, 1982) were obtained from the Arabidopsis Biological Resource Center and were cultured as described previously (Roeurn et al, 2016). The seeds were surfaced sterilized and sown on 1/2 MS basal medium with Gamborg's vitamins and 10 g L −1 sucrose (0.8% agar), and were vernalized at 4 °C for 2-3 d before germinating at 22 °C under constant light with a light intensity of ca.…”

“…The plants were cultured as described previously (Roeurn et al, 2016). Seeds were surfacesterilized in sodium hypochlorite solution and sown on MS basal medium with Gamborg's vitamins and 30 g L −1 sucrose (0.7% Agar).…”

“…In our previous study, using a cDNA based-suppression subtractive hybridization (SSH), we have identified genes differentially expressed between the wild type and EBC-less mutant, and have shown that a SSH-derived clone WM28, a putative jasmonate-induced gene, positively regulates trichome number in Arabidopsis by increase of the expression of genes for the components of trichome development-acting complex such as GL1 and GL3 (Roeurn et al, 2016). Here, we elucidated the other genetic determinants of EBC development in the common ice plant by analyzing the expression of the common ice plant homologs of genes known to be involved in the development of cotton fibers and Arabidopsis trichomes.…”