Characterization of VuMATE1 Expression in Response to Iron Nutrition and Aluminum Stress Reveals Adaptation of Rice Bean (Vigna umbellata) to Acid Soils through Cis Regulation

Abstract

Rice bean (Vigna umbellata) VuMATE1 appears to be constitutively expressed at vascular system but root apex, and Al stress extends its expression to root apex. Whether VuMATE1 participates in both Al tolerance and Fe nutrition, and how VuMATE1 expression is regulated is of great interest. In this study, the role of VuMATE1 in Fe nutrition was characterized through in planta complementation assays. The transcriptional regulation of VuMATE1 was investigated through promoter analysis and promoter-GUS reporter assays. The results showed that the expression of VuMATE1 was regulated by Al stress but not Fe status. Complementation of frd3-1 with VuMATE1 under VuMATE1 promoter could not restore phenotype, but restored with 35SCaMV promoter. Immunostaining of VuMATE1 revealed abnormal localization of VuMATE1 in vasculature. In planta GUS reporter assay identified Al-responsive cis-acting elements resided between -1228 and -574 bp. Promoter analysis revealed several cis-acting elements, but transcription is not simply regulated by one of these elements. We demonstrated that cis regulation of VuMATE1 expression is involved in Al tolerance mechanism, while not involved in Fe nutrition. These results reveal the evolution of VuMATE1 expression for better adaptation of rice bean to acid soils where Al stress imposed but Fe deficiency pressure released.

Keywords: Fe deficiency; acid soil; aluminum toxicity; cis-acting element; citrate secretion; evolution; transcriptional regulation.

FIGURE 1

Physiological and molecular responses of rice bean to Fe deficiency. Seedlings of rice bean were subject to nutrient solution with 20 μM Fe (+Fe) or without (-Fe) for 12 days. (A) SPAD value of newly expanded leaves. Data are means ± SD (n = 8). (B) Ferric chelate reductase (FCR) activity of roots. Data are means ± SD (n = 4). (C) RT-PCR analysis of the expression of VuMATE1 and VuIRT1 in both roots and shoots. (D) Quantitative real-time PCR analysis of the expression of VuIRT1 in roots. Data are means ± SD (n = 3). (E) Quantitative real-time PCR analysis of the expression of VuMATE1 in roots. Data are means ± SD (n = 3). Asterisks represent significant differences between +Fe and -Fe treatment at P < 0.05.

FIGURE 2

In planta complementation assay of VuMATE1 in Fe nutrition in Arabidopsis mutant frd3-1 driven by its native promoter (VuMATE1p::VuMATE1/frd3-1). (A) RT-PCR characterization of VuMATE1 expression in roots of two independent complemented lines (line 1 and line 2). (B) Phenotype analysis of leaf chlorosis in wild-type (WT), frd3-1, and two complemented lines. (C) Chlorophyll content of newly expanded leaves in wild-type (WT), frd3-1, and two complemented lines. Data are expressed as means ± SD (n = 4). Columns with different letters are significantly different at P < 0.05.

FIGURE 3

In planta complementation assay of VuMATE1 in Fe nutrition in Arabidopsis mutant frd3-1 driven by 35S CaMV promoter. (A) Phenotype analysis of leaf chlorosis in WT, frd3-1, and two transgenic lines overexpressing VuMATE1 (OX1 and OX2). (B) Root ferric precipitation of WT, frd3-1, and two transgenic lines overexpressing VuMATE1 (OX1 and OX2). Bar, 100 μm.

FIGURE 4

Cell-specificity of localization of VuMATE1 in roots. Immunostaining with anti-VuMATE1 antibody is shown in the root apex either before (A) or after Al stress for 9 h (B), and maturation root zone (C). Bar, 100 μm.

FIGURE 5

Citrate secretion from rice bean roots in response to Fe deficiency and Al stress. One-week-old seedlings were subject to nutrient solution with 20 μM Fe (+Fe) or without (-Fe) for 12 days after treatment, root exudates were collected in 0.5 mM CaCl₂ solution (pH 4.5) in the absence (-Al) or presence of 25 μM Al (+Al) for 6 h. Data are expressed as means ± SD (n = 4). Columns with different letters indicate significant difference at P < 0.05.

FIGURE 6

Histochemical analysis of GUS expression in transgenic Arabidopsis lines driven by different 5′ deletion sequence of VuMATE1 promoter. (A) Induction of VuMATE1 by Al in transgenic Arabidopsis carrying VuMATE1p::GUS constructs. The representative images show GUS staining in roots after 9 h of exposure to 0 (-Al) or 1 μM Al (+Al) activity in nutrient solution. (B) GUS expression in root apex and maturation root zone of transgenic Arabidopsis carrying the shortest promoter of VuMATE1 (-192 bp::GUS). At least two independent transgenic lines were used to analyze GUS expression. Bar, 100 μm.

FIGURE 7

VuMATE1p::GUS expression analysis in transgenic Arabidopsis plants. Transgenic seedlings were exposed to 10 μM Al, 10 μM ABA, or 10 μM SA for 9 h. Activation of the VuMATE1 promoter was observed by GUS staining (blue). At least two independent transgenic lines were used to analyze GUS expression. Bars, 100 μm.