Boron tolerance in barley is mediated by efflux of boron from the roots

Many plants are known to reduce the toxic effects of high soil boron (B) by reducing uptake of B, but no mechanism for limiting uptake has previously been identified. The B-tolerant cultivar of barley (Hordeum vulgare L.), Sahara, was shown to be able to maintain root B concentrations up to 50% lower than in the B-sensitive cultivar, Schooner. This translated into xylem concentrations that were approximately 64% lower and leaf concentrations 73% lower in the tolerant cultivar. In both cultivars, B accumulation was rapid and reached a steady-state concentration in roots within 3 h. In Schooner, this concentration was similar to the external medium, whereas in Sahara, the root concentration was maintained at a lower concentration. For this to occur, B must be actively extruded from the root in Sahara, and this is presumed to be the basis for B tolerance in barley. The extrusion mechanism was inhibited by sodium azide but not by treatment at low temperature. Several anion channel inhibitors were also effective in limiting extrusion, but it was not clear whether they acted directly or via metabolic inhibition. The ability of Sahara to maintain lower root B concentrations was constitutive and occurred across a wide range of B concentrations. This ability was lost at high pH, and both Schooner and Sahara then had similar root B concentrations. A predictive model that is consistent with the empirical results and explains the tolerance mechanism based on the presence of a borate anion efflux transporter in Sahara is presented.     

Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.     

Low temperature acclimation mediated by ethanol production is essential for chilling tolerance in rice roots

Rice seedlings (Oryza sativa L.) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in fatty acid composition in rice roots under the present experimental condition. Alcohol dehydrogenase (ADH) activity and ethanol concentration in the roots were increased with increasing duration of LT-PT up to 12 h, which indicates that LT-PT increased ethanol fermentation in the roots. 4-Methylpyrazole, a potent inhibitor of ADH, reduced the ethanol concentration and the chilling tolerance in the roots. This reduction of the chilling tolerance recovered with exogenously applied ethanol. Ethanol also induced 21- and 33-kD protein synthesis in the roots and these proteins may contribute the improvement of the tolerance. The present research suggests that LT-PT may increase chilling tolerance in rice roots owing to ethanol production, and ethanol may trigger a signal transduction cascade, which might lead to a decrease in membrane damage and injury.Key words: acclimation, alcohol dehydrogenase, chilling tolerance, ethanol, heat shock protein, low temperature, Oryza sativaAlcohol dehydrogenase (ADH; EC.1.1.1.1) gene and protein were induced by low temperature in Arabidopsis, maize and rice seedlings.^1,2,3 ADH is an enzyme involved in ethanolic fermentation and essential for plants to survive under anaerobic conditions.^4,5 However, it is unlikely that the induction of ADH by low temperture is due to a switch from aerobic respiration to anaerobic respiration as reported with anaerobic conditions.^2,6 Therefore, it is not clear that biological meanings of the induction of ADH in low temperature conditions.Rice seedlings (Oryza sativa L. cv. Nipponbare) were subjected to low temperature pretreatment (LT-PT; 10°C) for various length of time (1, 2, 4, 6, 12, 18, 24 h) followed by a 48-h chilling temperature stress (2°C). Chilling tolerance of rice roots was improved with increasing duration of LT-PT, but HT-PT longer than 12 h gave no additional improvement. LT-PT did not change in any fatty acid compositions in rice roots under the present experimental condition. Several plant species, such as oat, rye and spinach increased freezing tolerance due to the increasing unsaturation of fatty acids in plasma membranes, but this cold acclimation process required exposure of these plants to subzero temperature for 2–3 weeks.^7,8LT-PT increased ADH activity and ethanol concentration in rice roots, and the activity and the concentration were increased with increasing duration of LT-PT up to 12 h. Thus, LT-PT induced ethanolic fermentation system and stimulated ethanol production in the roots. 4-Methylpyrazole, which is a potent inhibitor of ADH and prevents ethanol production,⁹ reduced rice root growth to 40% of LP-PT root growth (Fig. 1), and the ethanol accumulation in the roots. This growth inhibition by 4-methylpyrazole recovered with exogenously applied ethanol. These results suggest that ethanol produced by LT-PT may contribute the chilling tolerance in the roots of the rice seedlings. In addition, an ADH deficient mutant of maize seedlings, which can not produce ethanol, was more sensitive to chilling temperature than their wild types.⁶Open in a separate windowFigure 1Effects of ethanol and 4-methylpyrazole on root growth of rice seedlings. Three-day-old rice seedlings were treated 12-h LT-PT (10°C) with or without 100 mM ethanol and/or 5 mM 4-methylpyrazole at 25°C for 24 h, and then subjected to chilling stress treatment (2°C, 48 h). Elongation of rice roots was determined over 48 h at 25°C after chilling stress treatment. Non-stressed seedlings and ethanol-treated seedlings were grown at 25°C. Chilling stressed seedlings were grown at 25°C for 24 h, and then subjected to chilling stress treatment. Means ± SE from five independent experiments with 20 plants for each determination are shown.When the seedlings were subjected to chilling temperature stress after ethanol treatment without LT-PT, the growth inhibition of rice roots by chilling temperature recovered from 22% to 71% of that of nonstressed roots (Fig. 1), which suggests that exogenously applied ethanol may improve chilling tolerance in the roots. It is also found that the ethanol treatment did not change in fatty acid composition in the roots at the temperature of this treatment (25°C).Chilling temperature induced lipid degradation in plant cells of cold-sensitive plants, such as cucumber, rice and soybean, as measured by an increase in malondialdehyde, which is a decomposition product of phospholipid peroxidation.¹⁰ Lipid peroxidation occurs when polyunsatured fatty acids are released from phospholipids by phospholipases and became substrates for lipoxygenases. Changes in the structural composition of the plasma membranes by lipid peroxidation cause the phase transition of the membrane from liquid to gel and the inactivation of membrane bound enzymes such as plasma membrane ATPase. Thus, the phase transition of the membranes was thought to be one of the primary causes of chilling injury.^11–13The addition of C₁ to C₆ alcohols including ethanol to model membranes increased fluidity of the membranes and lowered the phase transition temperature of the membranes.^14,15,16 Therefore, ethanol produced by LT-PT may prevent the phase transition of the membrane from liquid to gel, and lower the phase transition temperature of the membranes, which may contribute the acclimation to the chilling tolerance. In addition, ethanol induced an increase in ATPase activity in plasma membranes,⁶ and prevented chilling-induced ion leakage from plant tissues.¹⁷Ethanol is also known to stimulate the synthesis of heat shock protein (HSP) in yeast, bacteria and some other plants.^18,19 We thus determined the effect of ethanol on protein synthesis in rice roots by SDS-gel electrophoresis, and found that 21- and 33-kD protein synthesis were induced by ethanol. These proteins were also induced by heat shock treatment (45°C, 20 min). HSP was shown to be associated with the development of low temperature tolerance in spinach.^20,21 Thus, 21- and 33-kD proteins induced by ethanol may contribute the improvement of the chilling tolerance.The present research suggests that LT-PT-induced chilling tolerance may be owing to ethanol accumulation in rice roots. Accumulated ethanol may increase the fluidity of plasma membranes and lower the phase transition temperature of the membranes, and may also induce protein synthesis. This hypothesis is supported by exogenously applied ethanol which increased the chilling tolerance. Thus, ethanol might trigger a signal transduction cascade, which would lead to a decrease in membrane damage and injury. Further work needs to be done to test this possibility.     

Girdling affects carbohydrate-related gene expression in leaves,bark and roots of alternate-bearing citrus trees

Effects of girdling on carbohydrate status and carbohydrate-related gene expression in citrus trees were investigated. Alternate-bearing 'Murcott' (a Citrus reticulata hybrid of unknown origin) trees were girdled during autumn (25 Sep. 2001) and examined 10 weeks later. Girdling brought about carbohydrate (soluble sugar and starch) accumulation in leaves and shoot bark above the girdle, in trees during their fruitless, 'off' year. Trees during their heavy fruit load, 'on' year did not accumulate carbohydrates above the girdle due to the high demand for carbohydrates by the developing fruit. Girdling caused a strong decline in soluble sugar and starch concentrations in organs below the girdle (roots), in both 'on' and 'off' trees. Expression of STPH-L and STPH-H (two isoforms of starch phosphorylase), Agps (ADP-glucose pyrophosphorylase, small subunit), AATP (plastidic ADP/ATP transporter), PGM-C (phosphoglucomutase) and CitSuS1 (sucrose synthase), all of which are associated with starch accumulation, was studied. It was found that gene expression is related to starch accumulation in all 'off' tree organs. RNA levels of all the genes examined were high in leaves and bark that accumulated high concentrations of starch, and low in roots with declining starch concentrations. It may be hypothesized that changes in specific sugars signal the up- and down-regulation of genes involved in starch synthesis.     

Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus.

Shiga-like toxin is an iron-regulated cytotoxin quite similar to Shiga toxin from Shigella dysenteriae 1. The structural genes for Shiga-like toxin in Escherichia coli (sltA and sltB) appear to be transcribed as an operon from a promoter upstream of sltA. We used a gene fusion between the promoter and proximal portion of sltA with the gene for bacterial alkaline phosphatase to assess the regulation of toxin expression. Growth in low-iron conditions resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the presence of a null mutation in the fur locus, however, alkaline phosphatase activity was constitutively high regardless of the iron concentration. These data indicate negative regulation of the slt operon by the fur gene product. We used deletion analysis of the region upstream of the gene fusion to localize the promoter of the slt operon and to show that a region of DNA between the -35 and -10 boxes is necessary for iron regulation of slt expression. In this region, there is a 21-base-pair dyad repeat that is homologous to similar dyads in the promoter regions of three other fur-regulated genes. This region of dyad symmetry may represent an operator binding site for the Fur protein in the presence of iron.     

Light-regulated expression of the pea plastocyanin gene is mediated by elements within the transcribed region of the gene

Expression of the pea plastocyanin gene ( PetE ) is regulated by light in both pea and transgenic tobacco plants. However, the PetE promoter with the 5' untranslated leader region does not direct light-regulated expression of the GUS reporter gene in transgenic tobacco. This suggested that sequences downstream of the translation start of the PetE gene are required for light-regulated expression. To investigate this possibility the expression of a series of chimeric gene constructs in transgenic tobacco plants was examined to assess the contributions of the promoter, the 5' untranslated leader region, the coding region and the 3' region of the PetE gene to light-regulated expression. Both the coding region and the 5' untranslated leader region of the PetE gene were found to be required for full light regulation. Full light regulation of chimeric gene constructs containing the cauliflower mosaic virus (CaMV) 35S promoter required the deletion of CaMV 5' leader and polylinker sequences from the constructs. The presence of CaMV and polylinker sequences at the 5' end of the PetE leader masked the light regulation directed by the transcribed region of the pea PetE gene.