Genome-wide screening of salt tolerant genes by activation-tagging using dedifferentiated calli of Arabidopsis and its application to finding gene for Myo-inositol-1-p-synthase.

Aftab Ahmad,Shingo Goto,Sohei Ito,Yumiko Usui,Yasuo Niwa,Hirokazu Kobayashi,Kyoko Kobayashi,Tsutomu Nakayama,Masanori Shimizu,Zhong-Hua Chen

doi:10.1371/journal.pone.0115502

Abstract

Salinity represents a major abiotic stress factor that can adversely limit the production, quality and geographical distribution of crops. In this study we focused on dedifferentiated calli with fundamental cell functions, the salt tolerance of which had not been previously examined. The experimental approach was based on activation tagging without regeneration of plants for the identification of salt-tolerant mutants of Arabidopsis. Among 62,000 transformed calli that were screened, 18 potential mutants resistant to 150 mM NaCl were obtained. Thermal asymmetric interlaced (TAIL)-PCR was performed to determine the location of T-DNA integration in the genome. In one line, referred to as salt tolerant callus 1 (stc1), expression of a gene [At4g39800: myo-inositol-1-P-synthase 1 (MIPS1)] was considerably enhanced in calli. Plants regenerated from calli showed tolerance to salt in germination and subsequent growth. Retransformation of wild-type Arabidopsis with MIPS1 conferred salt tolerance, indicating that MIPS1 is the causal gene. The over-expression of MIPS1 increased the content of total inositol. The involvement of MIPS1 in salt tolerance through the fundamental cell growth has been proved in Arabidopsis.

Highlights

Salinity represents a major abiotic stress factor that imposes serious threats to agricultural industries worldwide
In an effort to investigate salt tolerance at the cellular level, roots of Arabidopsis were infected with Agrobacterium tumefaciens harboring the binary vector pRi35ADEn4 [15], which contained four copies of a 339-bp long cauliflower mosaic virus (CaMV) 35S enhancer [12] at the right border of the T-DNA and a gene for acetolactate synthase as a selectable marker [25]
It was observed that the survival and proliferation rates of mutant calli were significantly higher at different NaCl stress levels compared with untransformed calli (Fig 1), while they grew to the wild-type without salt stress as described below together with phenotypes of a myo-inositol-1-P-synthase 1 (MIPS1)-knockout (KO) line

Summary

Introduction

Salinity represents a major abiotic stress factor that imposes serious threats to agricultural industries worldwide. Over-expression of inositol-3-phosphate synthase from Spirodela polyrrhiza (SpMIPS), which resulted in up to a 4-fold increase in free inositol levels, was less associated with an increasing salt tolerance in Arabidopsis [23], it was argued that expression was not high enough to show significant results In addition to these reports concerning enhancement of salt tolerance resulting from the overexpression of MIPS homologs, similar effects were observed in transgenic tobacco and Arabidopsis plants following the overproduction of mannitol [59, 60]. When we compared the protein sequence of AtMIPS1 with those of PcMIPS, OsMIPS and SpMIPS, we found that AtMIPS1 possessed amino acid sequences highly identical to those of SpMIPS and OsMIPS, it was less homologous especially in the region consisting of Trp174-Ser210 in PcMIPS (Fig 9), which reportedly is responsible for salt tolerance [43]. In our search for identical local structures in AtMIPS1 and PcMIPS which make plants salt tolerant, a putative myristoylation site composed of GGKNTI at 410–415 in AtMIPS1 was found, leading to the speculation that it may play a role in the regulation of intercellular localization of MIPS or its interaction with some specific protein(s)

Methods

Results

Conclusion