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

Siranet Roeurn,Narihiro Hoshino,Ken-Taro Soejima,Yuuka Inoue,John C Cushman,Sakae Agarie

doi:10.1080/1343943x.2016.1221320

Siranet Roeurn, Narihiro Hoshino + Show 4 more

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https://doi.org/10.1080/1343943x.2016.1221320

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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.

Highlights

Salinity stress imposes two major physiological stresses on plants: osmotic stress and ionic injury
14 genes of unknown functions, whose roles might be related to epidermal bladder cells (EBC) development in the common ice plant, were subsequently subject to northern blot analysis
The differential mRNA of 12 genes of unknown function was confirmed and subsequently reconfirmed using reverse transcription-PCR (RT-PCR) expression analysis. These genes were categorized into two groups: genes whose transcripts were highly expressed in EBC-less mutant and genes whose transcripts were highly expressed in wild-type plants

Summary

Introduction

Salinity stress imposes two major physiological stresses on plants: osmotic stress and ionic injury. Osmotic stress is related to the adverse effect of excess salt on water status of a plant. High external ionic concentrations disrupt ion homeostasis, leading to the excessive accumulation of toxic ions such as Na+ and Cl− (Nui et al, 1995), and have adverse effects on cell membrane integrity, enzyme activities, nutrient uptake, and photosynthesis (Tester & Davenport, 2003). Halophytes have developed numerous strategies for adaptation to growth under high salinity concentrations, including osmotic adjustment through compartmentation of toxic and excess ions into epidermal bladder cells (EBCs), accumulation of compatible solutes, succulence, and secretion via salt-accumulating organs, such as salt glands (Flowers & Colmer, 2008; Shabala, 2013). In addition to sodium sequestration, EBCs likely serve as water reservoirs or as a secondary epidermis to reduce leaf water evaporation and prevent excessive UV damages (Adams et al, 1998)

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