Tonoplast- and Plasma Membrane-Localized Aquaporin-Family Transporters in Blue Hydrangea Sepals of Aluminum Hyperaccumulating Plant

Takashi Negishi,Kumi Yoshida,Masatake Kanai,Shoji Mano,Masahira Hattori,Mikio Nishimura,Kenshiro Oshima,Minami Matsui

doi:10.1371/journal.pone.0043189

Abstract

Hydrangea (Hydrangea macrophylla) is tolerant of acidic soils in which toxicity generally arises from the presence of the soluble aluminum (Al) ion. When hydrangea is cultivated in acidic soil, its resulting blue sepal color is caused by the Al complex formation of anthocyanin. The concentration of vacuolar Al in blue sepal cells can reach levels in excess of approximately 15 mM, suggesting the existence of an Al-transport and/or storage system. However, until now, no Al transporter has been identified in Al hyperaccumulating plants, animals or microorganisms. To identify the transporter being responsible for Al hyperaccumulation, we prepared a cDNA library from blue sepals according to the sepal maturation stage, and then selected candidate genes using a microarray analysis and an in silico study. Here, we identified the vacuolar and plasma membrane-localized Al transporters genes vacuolar Al transporter (VALT) and plasma membrane Al transporter 1 (PALT1), respectively, which are both members of the aquaporin family. The localization of each protein was confirmed by the transient co-expression of the genes. Reverse transcription-PCR and immunoblotting results indicated that VALT and PALT1 are highly expressed in sepal tissue. The overexpression of VALT and PALT1 in Arabidopsis thaliana conferred Al-tolerance and Al-sensitivity, respectively.

Highlights

The colors of Hydrangea macrophylla sepals vary from blue to purple and red, and it is well known that their color changes with different cultivation conditions and/or with transplanting [1,2]
To identify Al transporter genes from H. macrophylla, we planned and implemented the following strategy: 1) the preparation and sequencing of a full-length cDNA library from blue sepal tissue, 2) the generation of a custom microarray that was based on these cDNAs, 3) the selection of candidate genes on a microarray and 4) the implementation of a tolerance assay using an Al-sensitive yeast strain
To evaluate the reliability of the gene expression in this microarray, the genes encompassing the steps from chalcone synthase (CHS) to anthocyanidin 3-Oglucosyltransferase (3GT) in the anthocyanin biosynthetic pathway were analyzed, and the expected expression levels were determined for almost all of these genes, with the exception of chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H)

Summary

Introduction

The colors of Hydrangea macrophylla sepals vary from blue to purple and red, and it is well known that their color changes with different cultivation conditions and/or with transplanting [1,2]. To identify Al transporter genes from H. macrophylla, we planned and implemented the following strategy: 1) the preparation and sequencing of a full-length cDNA library from blue sepal tissue, 2) the generation of a custom microarray that was based on these cDNAs, 3) the selection of candidate genes on a microarray and 4) the implementation of a tolerance assay using an Al-sensitive yeast strain.

Results

Conclusion