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J Exp Bot
2010 Mar 01;615:1455-67. doi: 10.1093/jxb/erq023.
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HvALMT1 from barley is involved in the transport of organic anions.
Gruber BD
,
Ryan PR
,
Richardson AE
,
Tyerman SD
,
Ramesh S
,
Hebb DM
,
Howitt SM
,
Delhaize E
.
Abstract
Members of the ALMT gene family contribute to the Al(3+) resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al(3+) resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al(3+) resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [(14)C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells.
Fig. 1. Chromosomal localization of HvALMT1 in wheat–barley addition lines by PCR. PCR was performed with (a) HvALMT1-specific (qRTPCR-1 and qRTPCR-2; 514 bp product) and (b) TaALMT1-specific (TaALMT1R and TaALMT1F; 107 bp product) primers on genomic DNA of wheat cultivar Chinese Spring (CS), barley cultivar Betzes (B) (both parents of the wheat–barley addition lines), and wheat–barley addition lines containing barley chromosomes 1–7. The 100 bp and 500 bp bands are indicated for the DNA size markers (leftmost lanes).
Fig. 2. The effect of Al3+ exposure or P deficiency on HvALMT1 expression in shoots and roots of barley. HvALMT1 expression was determined using qRTPCR in plants of an Al3+-sensitive (Golden Promise) or Al3+-resistant (Dayton) cultivar grown for 4 d in hydroponics containing no added Al3+ (minus Al) or 2.5 μM Al3+ (plus Al); or for the P experiment in 13-day-old plants after 6 d of growth in hydroponics containing no added P (minus P) or 100 μM P (plus P). The expression level is shown relative to the internal reference gene GAPDH (mean ± SEM; n=3 or 4). For each panel, columns designated with different letters are significantly different (P <0.05; ns=not significant).
Fig. 3. Localization of GFP fluorescence in plants transformed with either a HvALMT1 promoter::GFP or a HvALMT1 protein::GFP reporter fusion. (A and B) The stomata of barley transformed with a HvALMT1 promoter::GFP fusion. (C and D) The stomata of wild-type barley. In E–H the upper and lower roots are of two lines of barley independently transformed with the HvALMT1 promoter::GFP fusion while the middle root is that of wild-type barley. (I and J) A transverse section through the root of barley transformed with the HvALMT1 promoter::GFP fusion. (K and L) A transverse section through the root of wild-type barley. I and K used the same image acquisition and manipulation settings. (M and O) The subcellular localization of fluorescence in leek epidermal cells transiently expressing a fusion of GFP with the C-terminus of HvALMT1. (N and P) The corresponding brightfield images. GFP fluorescence from the control (pPSUbiGFP) is shown in Q and R. The nucleus is visible within M, N, and Q. The gold particles used for the transformation of cells are marked in N by the arrow. Within O the position of the plasma membrane is marked by the white arrow and the position of two vesicles with GFP fluorescence is marked by the yellow arrows. In P the positions of the same two vesicles are marked with yellow arrows and the tonoplast is marked by the black arrow.
Fig. 4. Electrophysiology of Xenopus oocytes expressing HvALMT1. (a) Family of current curves measured in Xenopus laevis oocytes injected with water (control) or HvALMT1 cRNA in response to 500 ms voltage pulses between –140 mV and 60 mV in 20 mV steps. The data were collected with either 10 mM MES–BTP or 10 mM malate–BTP in the bathing solution (pH 4.5). Current–voltage curves generated from the currents measured in control oocytes (b) or HvALMT1-expressing oocytes (c) with different anions in the bathing solution. Currents were recorded from oocytes incubated in MES–BTP (MES), chloride–BTP (Chloride), malate–BTP (Malate), citrate–BTP (Citrate), or fumarate–BTP (Fumarate) at pH 4.5. Error bars show the SEM, n=6.
Fig. 5. Effect of pH on currents in Xenopus oocytes expressing HvALMT1. Xenopus laevis oocytes injected with HvALMT1 cRNA or water as a control were incubated overnight in 10 mM malate–BTP. The currents across the oocyte membrane were recorded using the two-electrode voltage clamp technique, while the oocytes were bathed in either 10 mM MES–BTP (MES), 10 mM chloride–BTP (Cl), or 10 mM malate–BTP (malate) at external pH 4.5, 5.5, or 7.5. Shown is the current across the membrane of oocytes clamped at 60 mV and –140 mV. Error bars show the SEM (n=3).
Fig. 6. Efflux of radioactively labelled malate from Xenopus oocytes expressing HvALMT1. Oocytes were pre-loaded with [14C]malate and the radioactivity of the bathing solution was measured. Oocytes were injected with either HvALMT1 cRNA or water as a control and incubated at external pH 7.5 or pH 4.5. Shown is the mean efflux of radioactive malate from the oocyte 2 min and 5 min after injection. Error bars show the SEM (n=5), and columns with different letters are significantly different (P <0.05; n=5).
Fig. 7. Uptake of radioactively labelled malate by Xenopus oocytes. Oocytes injected with HvALMT1 cRNA or water as a control were incubated in [14C]malate at external pH 7.5 or pH 4.5 for 30 min and then digested in acid to determine the rate of malate uptake. Error bars show the SEM (n=5), and columns with different letters are significantly different (P <0.05; n=5).
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