Transposon-Mediated Alteration of TaMATE1B Expression in Wheat Confers Constitutive Citrate Efflux from Root Apices

The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3′ regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance.Intraspecific variation in aluminum (Al3+) tolerance is evident in many crop species and has been shown to be under either simple or complex genetic control (Ma et al., 2004; Magalhaes et al., 2007; Krill et al., 2010; Famoso et al., 2011). In wheat (Triticum aestivum), Al3+ tolerance is primarily associated with efflux of organic anions from root tips, with at least two independent mechanisms being involved. The first and most widely distributed among genotypes is the Al3+-activated efflux of malate from root apices (Delhaize et al., 1993, Ryan et al., 1995). More recently, a second and less prevalent mechanism has been identified that relies on the constitutive efflux of citrate from root apices (Ryan et al., 2009).Organic anions protect roots by chelating and detoxifying Al3+ in the apoplast and rhizosphere around sensitive root apices (Delhaize et al., 1993, 2012; Ryan et al., 2001). For example, Al3+-tolerant genotypes of wheat release significantly more malate than sensitive genotypes (Ryan et al., 1995). The efflux of malate from root apices is mediated by the TaALMT1 (for Al3+-activated malate transporter) gene, which resides on chromosome 4DL (Raman et al., 2005, 2008; Zhou et al., 2007). TaALMT1 encodes an Al3+-activated anion channel permeable to malate located on the plasma membrane of root cells (Sasaki et al., 2004; Yamaguchi et al., 2005; Piñeros et al., 2008; Zhang et al., 2008). Similarly, ALMT1 genes contribute to Al3+ tolerance in Arabidopsis (Arabidopsis thaliana), oilseed rape (Brassica napus), and rye (Secale cereale; Ryan et al., 2011). While all members of the ALMT family characterized to date encode transport proteins, only a minority are involved in Al3+ tolerance. The others contribute to physiological processes related to ionic relations and osmotic adjustment (Kovermann et al., 2007; Gruber et al., 2010; Meyer et al., 2010, 2011; Sasaki et al., 2010).In many plant species, citrate efflux from root apices also confers Al3+ tolerance. The genes controlling citrate efflux were first identified in sorghum (Sorghum bicolor; SbMATE [for multidrug and toxic compound extrusion]; Magalhaes et al., 2007) and barley (Hordeum vulgare; HvAACT1; Furukawa et al., 2007), with additional genes subsequently identified in Arabidopsis (Liu et al., 2009), maize (Zea mays; Maron et al., 2010), rice (Yokosho et al., 2009), and rice bean (Vigna umbellate; Yang et al., 2011). These genes belong to a large and ubiquitous family of MATE genes that were first identified in prokaryotes (Hvorup et al., 2003). SbMATE and HvAACT1 are part of a subset of plant MATE genes that facilitate citrate efflux (Liu et al., 2009; Magalhaes, 2010) with roles in Al3+ tolerance and iron (Fe) nutrition. For example, FRD3 from Arabidopsis and OsFRDL1 from rice both encode MATE proteins located in vascular tissue, where they release citrate into the xylem to enable Fe transport to shoots (Durrett et al., 2007). Recent evidence suggests that the Al3+ tolerance gene in barley, HvAACT1, was coopted from an original function in Fe nutrition by a random mutation. Al3+-tolerant genotypes of barley have a 1-kb insertion in the 5′ untranslated region (UTR) of the HvAACT1 coding region that alters its expression pattern. The insertion extends HvAACT1 expression to the root apices, which, in the presence of Al3+, causes citrate efflux from the apices and enhanced Al3+ tolerance (Fujii et al., 2012).Evidence that citrate efflux confers Al3+ tolerance in some genotypes of wheat was provided by Ryan et al. (2009). The trait was mapped to the long arm of chromosome 4B and cosegregated with an EST showing 94% sequence identity to HvAACT1. This EST was expressed only in the root apices of wheat lines that showed citrate efflux (e.g. cv Carazinho; Ryan et al., 2009), suggesting that a MATE gene encodes citrate efflux from root apices. Here, we describe the isolation and characterization of a MATE gene from wheat named TaMATE1B that encodes a citrate transporter located on the plasma membrane. A large transposable element-like sequence was found to be inserted near the start of the TaMATE1B coding region specifically in genotypes that release citrate. We demonstrate that the fragment functions as a promoter that extends TaMATE1B expression to root apices and discuss how this mutation recruited a gene to confer a new phenotype associated with Al3+ tolerance.     

Chloride transport and compartmentation within main and lateral roots of two grapevine rootstocks differing in salt tolerance

Root Cl^? transport was investigated using ³⁶Cl^? flux analysis in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance; 1103 Paulsen (salt-tolerant) and K 51–40 (salt sensitive). Initial ³⁶Cl^? influx to the root was greater in Paulsen than K 51–40. This flux, attributed to the Cl^? influx to the cytoplasm (Φ _oc) increased with increasing external concentrations of Cl^? for plants adapted to growth in 30 mM NaCl. The concentration kinetics in this high concentration range could be fit to a Michaeils–Menton equation. There was no significant difference between genotypes in Km (28.68 ± 15.76 and 24.27 ± 18.51 mM for Paulsen and K 51–40, respectively), but Paulsen had greater V _max (0.127 ± 0.042) compared to K 51–40 (0.059 ± 0.026 μm g^?1 FW min^?1). In Paulsen, the main root had greater contribution to ³⁶Cl^? uptake than lateral roots, there being no significant difference in lateral root influx between the genotypes. ³⁶Cl^? transport to the shoot of K 51–40 was greater than for Paulsen. It was estimated that efflux rate from the xylem parenchyma cells to the xylem vessels (Φ _cx) in K 51–40 was twice that of Paulsen. Compartmental analysis from ³⁶Cl^? efflux kinetics confirmed the larger Φ _oc and the higher ratio of main to lateral root Φ _oc for Paulsen. Efflux from the cytoplasm (Φ _co) was higher than 95 % of Φ _oc indicating a high degree of cycling across the plasma membrane in roots at these high external Cl^? concentrations. Paulsen appears to keep the cytoplasmic Cl^? concentration in roots lower than K 51–40 via greater efflux to the vacuole and to the outside medium. The difference in salt tolerance between the genotypes can be attributed to different Cl^? transport properties at the plasma membrane and tonoplast and particularly in Cl^? efflux to the xylem.     

Sources of water used by riparian Eucalyptus camaldulensis overlying highly saline groundwater

Water sources of Eucalyptus camaldulensis Dehn. trees were investigated on a semiarid floodplain in south-eastern Australia. The trees investigated ranged in distance from 0.5 to 40 m from a stream, with electrical conductivity 0.8 dSm^–1, and grew over groundwater with electrical conductivity ranging from 30 to 50 dSm^–1. The sources of water being used by the trees were investigated using the naturally occurring stable isotopes of water and measurements of soil water potential. Xylem water potential and leaf conductance were also examined to identify the trees' response to using these sources of water. Trees at distances greater than about 15 m from the stream used no stream water. The trees used groundwater in summer and a combination of groundwater and rain-derived surface-soil water (0.05–0.15 m depth) in winter. In doing so they suffered water stress at electrical conductivities higher than approximately 40 dSm^–1 (equivalent to approximately –1.4 MPa). Trees adjacent to the stream used stream water directly in summer, but may have used stream water from the soil profile in winter, after the stream had risen and recharged the soil water. E. camaldulensis appeared to be partially opportunistic in the sources of water they used.     

Inward membrane current inChara inflata: I. A voltage- and time-dependent Cl− component

Summary An inward current which increases in magnitude over a period of seconds is activated when the membrane ofChara inflata (a green alga) in a K⁺-conductive state is hyperpolarized by a voltage clamp. The peak current and the half-time of activation are exponentially dependent on membrane potential difference. It was found by using an external Cl^– electrode that the component exponentially dependent on potential was due to an efflux of Cl^–. The measured current-voltage curves and the kinetics of deactivation of the current showed that other time-dependent components contributed to the net inward current. The punchthrough theory of Coster (Biophys. J. 5:669–686, 1965) does not adequately explain the inward current since a punchthrough potential could not be obtained, and the inward current was distinctly time dependent. The voltage and time dependence of the inward current strongly suggests that the Cl^– efflux activated by hyperpolarization is through voltage-gated channels which open more frequently as the membrane is hyperpolarized.     

Reduction of Visceral Adipose Tissue and Improvement of Metabolic Indices: Effect of Dexfenfluramine in NIDDM

Increased visceral adipose tissue is thought to contribute to impaired glucose tolerance. We studied 10 men with non-insulin dependent diabetes (NIDDM) before and after a 12-week intervention study using dexfenfluramine. Subjects had a mean body mass index (BMI) of 26.4 ± 1.7 kg\m² and had an abdominal distribution of body fatness (waist-to hip ratio >0.9). Anthropometric indices, biochemistry, macronutrient intake from 7-day food records as well as a euglycaemic glucose clamp and magnetic resonance imaging (MRI) were performed at week 0 and week 12. Abdominal adipose tissue area measured by MRI was reduced from 854 ± 270 cm² to 666 ± 231 cm² (p=0.003) due mainly to a selective 32% reduction in visceral fat area from 484 ± 230 cm² to 333 ± 72 cm² (p=0.002). Insulin sensitivity improved from 0.29 ± 0.13 [min^?1 (mU/L)] to 0.54 ± 0.21 [min^?1 (mU/L)] (p=0.01) and C-peptide levels reduced from 0.77 ± 0.24 μmol/L to 0.58 ± 0.15 μmol/L (p=0.002). The reductions in fasting glucose and glycated haemoglobin failed to achieve significance. Fasting total cholesterol and triglyceride levels significantly reduced (p=<0.001 and p=0.021 respectively). There was a reduction in total energy intake (p=0.005) due to a significant reduction in calories obtained from fat (p<0.001). Thus dexfenfluramine was shown to be a useful adjunct therapy for the reduction of visceral fat in abdominally-obese men with NIDDM with an associated improvement in insulin sensitivity.     

Effect of low oxygen concentration on the electrical properties of cortical cells of wheat roots

Short-term effect of oxygen-deficiency on the membrane potential difference (PD), membrane resistance of cortical cells and electrical coupling between cortical cells was investigated using excised wheat roots. Hypoxia rapidly depolarised the membrane potential of the cortical cells by about 60 mV, while hypoxia had little effect on the membrane resistance of the cells. No significant change in membrane resistance by potassium channel blockers, TEA+ and verapamil, under hypoxia was observed. The electrical coupling ratio, which is a measure of plasmodesmatal resistance, between cortical cells of wheat roots was 5.9 % in aerated solution and was not affected by the low oxygen treatment, suggesting that solute transport through cytoplasmic annulus of plasmodesmata could not be affected. The possible involvement of the endoplasmic reticulum in intercellular transport of solute and water is discussed.     

Water Relations of Seagrasses: STATIONARY VOLUMETRIC ELASTIC MODULUS AND OSMOTIC PRESSURE OF THE LEAF CELLS OF HALOPHILA OVALIS, ZOSTERA CAPRICORNI, AND POSIDONIA AUSTRALIS

The stationary volumetric elastic modulus (ε_s) of the leaf cells of three seagrasses (Halophila ovalis (R.Br.) Hook, Zostera capricorni Aschers, and Posidonia australis Hook f.) was evaluated from estimates of ε_s plus intracellular osmotic pressure (ε_s + II_i) and II_i. The estimates of (ε_s + II_i) were made using a linear displacement transducer to measure very small changes in thickness of leaf tissue produced by changes in external osmotic pressure (II_o). ε_s increases with increasing turgor pressure in each of the species and the maximum values of ε_s are: 22 megapascals for H. ovalis, 17 megapascals for Z. capricorni, and 51 megapascals for P. australis.     

Floodwater infiltration through root channels on a sodic clay floodplain and the influence on a local tree species Eucalyptus largiflorens

Dieback of riparian species on floodplains has been attributed to increased soil salinisation due to raised groundwater levels, resulting from irrigation and river regulation. This is exacerbated by a reduction in flooding frequency and duration of inundation. For the Chowilla floodplain on the River Murray raised water tables have increased the amount of salts mobilised in the soil profile, causing the trees to experience salt induced water stress. For the trees to survive in the long term, salts need to be leached from the root zone.This study investigated whether floodwater infiltrates through channels created by E. largiflorens (black box) roots, flushing salts away from roots, thereby allowing the trees to increase their water uptake. Trees at different sites on the floodplain were artificially flooded, by pumping 1.5 kL of creek water into impoundments constructed around the trees. Gas exchange parameters, and pre-dawn and midday water potential were measured the day before, the day after and one week after the artificial flood and compared against trees that were not flooded. Pre-dawn and midday water potentials were also measured one month after the flood. After flooding, the trees experienced less water stress, indicated by an increase in water potential of less than 0.2 MPa, in comparison to non-flooded control trees. However, this response was not evident one month after flooding. The response to flooding did not result in increased rates of transpiration, stomatal conductance or photosynthesis, even though flooding effectively doubled the trees yearly water supply.The infiltration of floodwater in the impoundments around E. largiflorens was also compared to that of impoundments on bare ground. Floodwater infiltrated 2 – 17 times faster around trees than on adjacent bare ground, for parts of the floodplain not grazed by livestock. Tracer dye experiments indicated that bulk flow of water through pores down the profile was the reason for the enhanced infiltration. Flooding leached salts in direct vicinity of tree roots, but only leached small amounts of salts from the bulk soil.     

Unparallel diversification in bacterial microcosms

Adaptive speciation has gained popularity as a fundamental process underlying the generation of diversity. We tested whether populations respond to similar forms of disruptive selection by diversifying in similar or parallel ways by investigating diversified populations of Escherichia coli B evolved in glucose and glucose-acetate environments. In both environments, the populations have differentiated into two phenotypes, named for their characteristic colony morphologies: large (L) and small (S). Each type is heritable and this polymorphism (or 'diversified pair') appears to be maintained by negative frequency dependence. The L and S phenotypes from different environments are convergent in their colony morphology and growth characteristics. We tested whether diversification was parallel by conducting competition experiments between L and S types from different environments. Our results indicate that replicate diversified pairs from different environments have not diversified in parallel ways and suggest that subtle differences in evolutionary environment can crucially affect the outcome of adaptive diversification.