Plasma membrane aquaporins of the PIP1 and PIP2 subfamilies facilitate hydrogen peroxide diffusion into plant roots.

BACKGROUND: The permeability of plasma membrane aquaporins (PIPs) to small solutes other than water greatly diversifies their potential functions in plant development and metabolic processes. One such process is stress signalling in which hydrogen peroxide (H2O2) plays a major role. Based on transport assays carried out in yeast, there are differences in the degree to which PIPs of Arabidopsis thaliana, are permeable to H2O2 and thus they may differentially facilitate transmembrane diffusion. Here, we test whether specific PIPs aid in the transmembrane diffusion of H2O2 to such an extent that knocking-out PIPs affects plant phenotype. We examined changes in growth and morphology, including biomass accumulation, root system architecture and relative water content, as well as gas exchange, across two H2O2 treatments in knockout mutants of A. thaliana. RESULTS: We could infer that PIP-type aquaporins are permeable to H2O2 in planta and that this permeability is physiologically relevant in a plant's response to oxidative stress. In particular, the lack of functional PIP2;3 confers resistance to exogenously applied H2O2 indicating that it facilitates H2O2 entry into root cells. Additionally, PIP1;1 and PIP2;6 were found to facilitate H2O2 diffusion, while PIP2;2 is required for proper root growth under controlled conditions. MAIN FINDINGS: We conclude that PIPs are physiologically relevant conduits for H2O2 diffusion in the A. thaliana roots and participate in the regulation of stress responses.

Microstructure and Mechanical Properties of Al-Si-Fe-Cu-Mn-x Zn Alloys Processed by Extrusion.

In this work, Al-0.15Si-0.2Fe-0.3Cu-0.9Mn alloys with different Zn addition (0, 0.15 and 0.3 wt%) were melted and extruded at 200 °C. The effect of Zn on the microstructure, texture evolution and mechanical properties of Al-0.15Si-0.2Fe-0.3Cu-0.9Mn alloys was investigated using scanning electron microscope (SEM), equipped with energy-dispersive X-ray spectrometry (EDS) and electron backscatter diffraction (EBSD) and in the present study. In order to evaluate the mechanical properties, we implemented the tensile tests by a universal material test machine. Al-0.15Si-0.2Fe- 0.3Cu-0.9Mn-xZ resulted in the formation of Al-(Fe, Mn)-Si and Al-(Fe, Mn) intermetallic compounds. The formation of the intermetallic compound and this phase was broken in to small particles during extrusion. The ultimate strength and elongation of the as-extruded Al-0.15Si-0.2Fe-0.3Cu- 0.9Mn alloy were 96.51 MPa and 34.01%, while those of the Al-0.15Si-0.2Fe-0.3Cu-0.9Mn-0.3Zn alloy were 99.08 MPa and 36.21%, respectively. Al-0.15Si-0.2Fe-0.3Cu-0.9Mn alloys with Zn addition resulted in improving the strength with no reduction in elongation.

Functionally redundant LNG3 and LNG4 genes regulate turgor-driven polar cell elongation through activation of XTH17 and XTH24.

KEY MESSAGE: In this work, we genetically characterized the function of Arabidopsis thaliana, LONGIFOLIA (LNG1), LNG2, LNG3, LNG4, their contribution to regulate vegetative architecture in plant. We used molecular and biophysical approaches to elucidate a gene function that regulates vegetative architecture, as revealed by the leaf phenotype and later effects on flowering patterns in Arabidopsis loss-of-function mutants. As a result, LNG genes play an important role in polar cell elongation by turgor pressure controlling the activation of XTH17 and XTH24. Plant vegetative architecture is related to important traits that later influence the floral architecture involved in seed production. Leaf morphology is the primary key trait to compose plant vegetative architecture. However, molecular mechanism on leaf shape determination is not fully understood even in the model plant A. thaliana. We previously showed that LONGIFOLIA (LNG1) and LONGIFOLIA2 (LNG2) genes regulate leaf morphology by promoting longitudinal cell elongation in Arabidopsis. In this study, we further characterized two homologs of LNG1, LNG3, and LNG4, using genetic, biophysical, and molecular approaches. Single loss-of-function mutants, lng3 and lng4, do not show any phenotypic difference, but mutants of lng quadruple (lngq), and lng1/2/3 and lng1/2/4 triples, display reduced leaf length, compared to wild type. Using the paradermal analysis, we conclude that the reduced leaf size of lngq is due to decreased cell elongation in the direction of longitudinal leaf growth, and not decreased cell proliferation. This data indicate that LNG1/2/3/4 are functionally redundant, and are involved in polar cell elongation in Arabidopsis leaf. Using a biophysical approach, we show that the LNGs contribute to maintain high turgor pressure, thus regulating turgor pressure-dependent polar cell elongation. In addition, gene expression analysis showed that LNGs positively regulate the expression of the cell wall modifying enzyme encoded by a multi-gene family, xyloglucan endotransglucosylase/hydrolase (XTH). Taking all of these together, we propose that LNG related genes play an important role in polar cell elongation by changing turgor pressure and controlling the activation of XTH17 and XTH24.

Evolution of Nanostructure Through Thickness of Deoxidized Low-Phosphorous Copper by Three-Layer Stack Accumulative Roll-Bonding.

A nanostructured deoxidized low-phosphorous copper (DLPC) was fabricated by three-layer stack accumulative roll-bonding process. Three sheets of 1 mm in thickness, 30 mm in width and 300 mm in length were stacked up and roll-bonded to thickness of 1 mm by two-pass cold rolling. The bonded sheet was cut in three pieces of same length, then stacked up and roll-bonded to the thickness of 1 mm again. The evolution of nanostructure through thickness with three-layer stack ARB were investigated in detail. It was found that the microstructure has been evolved from a dislocation cell structure to a nano grained structure with the proceeding of ARB cycles. The average grain thickness of 45 µm in initial decreased to 170 nm after 7 cycles of the ARB. The heterogeneity in microstructure through thickness was also largely decreased by the ARB. These results suggest that three-layer stack ARB is an effective process for a formation of nanostructure of DLPC alloy.

Characteristics of Nano Grained AA1050/AA5052 Al Sheets Fabricated by Accumulative Roll-Bonding.

Accumulative roll-bonding (ARB) is the most appropriate process for sheet-shaped materials because it can be carried out readily by utilizing the conventional rolling apparatus. In this study, a nanostructured AA1050/AA5052 Al alloy sheet was successfully fabricated by four-layer stack ARB process. The ARB of AA1050 and AA5052 alloy sheets was performed up to 6 cycles without a lubricant at ambient temperature. The sample fabricated by the ARB was a multi-layer aluminum alloy sheet in which AA1050 and AA5052 layers are alternately stacked. The layer thickness of the each alloy became thinner and elongated to the rolling direction with the number of ARB cycles. The grain size decreased with increasing of the number of ARB cycles, after 6 cycles it became about 180 nm in thickness. The fraction of high angle grain boundaries increased with the number of ARB cycles. The tensile strength also increased with the ARB, it reached 305 MPa which is about 2.1 times that of the as-received AA1050. The mechanical properties of a multi-layer AA1050/AA5052 alloy fabricated by the ARB were compared to those of the other materials.

Effect of Mn and AlTiB Addition and Heattreatment on the Microstructures and Mechanical Properties of Al-Si-Fe-Cu-Zr Alloy.

The microstructure and mechanical properties of as-extruded Al-0.1 wt%Si-0.2 wt%Fe- 0.4 wt%Cu-0.04 wt%Zr-xMn-xAlTiB (x = 1.0 wt%) alloys under various annealing processes were investigated and compared. After the as-cast billets were kept at 400 °C for 1 hr, hot extrusion was carried out with a reduction ratio of 38:1. In the case of the as-extruded Al-Si-Fe-Cu-Zr alloy at annealed at 620 °C, large equiaxed grain was observed. When the Mn content is 1.0 wt%, the phase exhibits a skeleton morphology, the phase formation in which Mn participated. Also, the volume fraction of the intermetallic compounds increased with Mn and AlTiB addition. For the Al-0.1Si-0.2Fe-0.4Cu-0.04Zr alloy with Mn and AlTiB addition from 1.0 wt%, the ultimate tensile strength increased from 100.47 to 119.41 to 110.49 MPa. The tensile strength of the as-extruded alloys improved with the addition of Mn and AlTiB due to the formation of Mn and AlTiB-containing intermetallic compounds.

Grain Refinement of Al-Si-Fe-Cu-Zn-Mn Based Alloy by Al-Ti-B Alloy and Its Effect on Mechanical Properties.

We investigated the effects of Al-5.0wt%Ti-1.0wt%B addition on the microstructure and mechanical properties of the as-extruded Al-0.15wt%Si-0.2wt%Fe-0.3wt%Cu-0.15wt%Zn-0.9wt%Mn based alloys. The Aluminum alloy melt was held at 800 °C and then poured into a mould at 200 °C. Aluminum alloys were hot-extruded into a rod that was 12 mm in thickness with a reduction ratio of 38:1. AlTiB addition to Al-0.15Si-0.2Fe-0.3Cu-0.15Zn-0.9Mn based alloys resulted in the formation of Al3Ti and TiB2 intermetallic compounds and grain refinement. With increasing of addition AlTiB, ultimate tensile strength increased from 93.38 to 99.02 to 100.01 MPa. The tensile strength of the as-extruded alloys was improved due to the formation of intermetallic compounds and grain refinement.

Mechanical Properties and Fracture Behaviors of the As-Extruded Mg-5Al-3Ca Alloys Containing Yttrium at Elevated Temperature.

Effects of yttrium (Y) addition on mechanical properties and fracture behaviors of the as-extruded Mg-Al-Ca based alloys at elevated temperature were investigated by a tensile test. After hot extrusion, the average grain size was refined by Y addition and eutectic phases were broken down into fine particles. Y addition to Mg-5Al-3Ca based alloy resulted in the improvement of strength and ductility at elevated temperature due to fine grain and suppression of grain growth by formation of thermally stable Al2Y intermetallic compound.

Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings.

The contribution of hyphae to water transport in ectomycorrhizal (ECM) white spruce (Picea glauca) seedlings was examined by altering expression of a major water-transporting aquaporin in Laccaria bicolor. Picea glauca was inoculated with wild-type (WT), mock transgenic or L. bicolor aquaporin JQ585595-overexpressing (OE) strains and exposed to root temperatures ranging from 5 to 20°C to examine the root water transport properties, physiological responses and plasma membrane intrinsic protein (PIP) expression in colonized plants. Mycorrhization increased shoot water potential, transpiration, net photosynthetic rates, root hydraulic conductivity and root cortical cell hydraulic conductivity in seedlings. At 20°C, OE plants had higher root hydraulic conductivity compared with WT plants and the increases were accompanied by higher expression of P. glauca PIP GQ03401_M18.1 in roots. In contrast to WT L. bicolor, the effects of OE fungi on root and root cortical cell hydraulic conductivities were abolished at 10 and 5°C in the absence of major changes in the examined transcript levels of P. glauca root PIPs. The results provide evidence for the importance of fungal aquaporins in root water transport of mycorrhizal plants. They also demonstrate links between hyphal water transport, root aquaporin expression and root water transport in ECM plants.

Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis.

The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 µm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.

Microstructure and texture of a nano-grained complex Al alloy fabricated by accumulative roll-bonding of dissimilar Al alloys.

An ultrafine grain (UFG) complex lamella aluminum alloy sheet was successfully fabricated by ARB process using AA1050 and AA6061. The lamella thickness of the alloy became thinner and elongated to the rolling direction with increasing the number of ARB cycles. By TEM observation, it is revealed that the aspect ratio of UFGs formed by ARB became smaller with increasing the number of ARB cycles. In addition, the effect of ARB process on the development of deformation texture at the quarter thickness of ARB-processed sheets was clarified. ARB process leaded to the formation of the rolling texture with shear texture and weak cube orientation. The subdivision of the grains to the rolling direction began to occur after 3 cycles of the ARB, resulting in formation of ultrafine grains with small aspect ratio. After 5 cycles, the ultrafine grained structure with the average grain diameter of 560 nm develops in almost whole regions of the sample.

Fabrication and evaluation of nanostructure Al-SiCp composite by accumulative roll-bonding.

A nanostructured Al/SiCp composite was fabricated by accumulative roll bonding (ARB) process. The amount of SiC particles introduced into aluminum sheet by one cycle ARB was 0.22 mg/cm2. Increment of the number of ARB cycles increased the amount of SiC particles and enhanced the homogeneity of the distribution of SiC particles. Microstructural change in aluminum matrix with proceeding of the ARB was very similar to that of the ARB-processed unreinforced aluminum; showing a dislocation cell and/or sub-grain structure in samples of lower ARB cycles and ultrafine grained structure in samples of higher ARB cycles. The variation of tensile strength with the ARB was also very similar to that of unreinforced aluminum. However, the effect of strengthening by the ARB was higher in the Al/SiCp composite than in unreinforced aluminum due to an existence of the SiC particles and the formation of finer grains.

Development of nano-grains in deoxidized low-phosphorous copper by accumulative roll-bonding process.

A deoxidized low-phosphorous copper (DLPC) was processed by accumulative roll-bonding (ARB). The evolution of nano-grains in DLPC with the ARB was investigated by TEM and EBSD analysis. TEM observation revealed that the ultrafine grains equiaxed to ND and elongated to RD developed in the sample after 8-cycle ARB. In addition, it was found that the mean spacing of grain boundaries, which was 45 microm in initial material, reduced to 5.2 microm after 1 cycle, 650 nm after 3 cycles, 510 nm after 5 cycles, and then surprisingly 190 nm after 8 cycles, respectively. In addition, the fraction of high-angle boundaries in the sample after 1-cycle ARB was 0.22, but it after 8-cycle ARB was surprisingly 0.70. Therefore, it is concluded that the ARB is a very effective process for development of ultrafine grains in case of DLPC as well.

Fabrication of nanostructured deoxidized low-phosphorous copper processed by three-layer stack accumulative roll-bonding.

A nanostructured deoxidized low-phosphorous copper (DLPC) was fabricated by three-layer stack accumulative roll-bonding (ARB) process. The microstructural evolution and the variation of mechanical properties with three-layer stack ARB were investigated in detail. It was found that the microstructure has been evolved from a dislocation cell structure to ultrafine grained structure as the number of ARB cycles increases. In addition, the mean spacing of grain boundaries, which was 45 microm in initial material, reduced to 2.1 microm after 1 cycle, 360 nm after 3 cycles, 250 nm after 5 cycles, then 170 nm after 7 cycles, progressively. The fraction of high-angle grain boundaries after 1-cycle ARB was no more than 0.27, but it increased with the number of ARB cycles, and became surprisingly more than 0.7 after 7-cycle ARB. The tensile strength increased with the number of ARB cycles, and then after 7 cycles it reached about 600 MPa, which is about 2.5 times higher than that of the initial material. Therefore, the three-layer stack ARB is very effective for development of ultrafine grains and high strengthening of DLPC alloy.

Electrical characteristics of printed Ag nanopaste on polyimide substrate.

We investigated the effects of sintering temperature on the microstructural evolution and electrical characteristics of screen-printed Ag patterns. A conducting paste containing 20 nm Ag nanoparticles (73 wt%) was screen printed onto a polyimide (PI) substrate and sintered at a temperature of 150, 200, 250 and 300 degrees C for 30 min. The microstructures of the sintered patterns were examined using field emission scanning electron microscopy (FESEM). The resistivity under the application of a DC signal decreased with increasing temperature. In the frequency range from 10 MHz to 20 GHz, the S-parameters of the sintered Ag conducting patterns were measured. The S-parameters indicated that the insertion losses at high frequency decreased with increasing sintering temperature due to the formation of interparticle necking after sintering.

Formation of Heterogeneous Microstructure in AA1050/AA5052/AA6061/AA1050 Layered Sheet Processed by Cold Roll-Bonding and Subsequent Annealing.

A cold roll-bonding process was applied to fabricate an AA1050/AA6061/AA5052/AA1050 four-layer clad sheet and subsequently annealed. Three types of aluminum alloy sheets such as AA1050, AA6061 and AA5052 with 2 mm thickness, 40 mm width and 300 mm length were stacked up each other after such surface treatment as degreasing and wire brushing, then reduced to a thickness of 2 mm by multi-pass cold rolling. The rolling was performed at ambient temperature without lubricant using a 2-high mill with a roll diameter of 400 mm at rolling speed of 6.0 m/sec. The roll bonded AA1050/AA6061/AA5052/AA1050 clad sheet was then annealed for 0.5 h at 200~400 °C. Microstructures of the as-roll bonded and subsequently annealed aluminum sheets are investigated by electron back scatter diffraction (EBSD) measurement. After rolling, the roll-bonded AA1050/AA5052/AA6061/AA1050 sheet showed a typical deformation structure that the grains are largely elongated to the rolling direction. However, after annealing, it exhibits a very heterogeneous structure consisting of both deformation structure and recrystallization structure containing nanometer order grains. The formation of this heterogeneous structure and texture with annealing is investigated in detail through EBSD analysis.

Role of aquaporins in root water transport of ectomycorrhizal jack pine (Pinus banksiana) seedlings exposed to NaCl and fluoride.

Effects of ectomycorrhizal (ECM) fungus Suillus tomentosus on water transport properties were studied in jack pine (Pinus banksiana) seedlings. The hydraulic conductivity of root cortical cells (L(pc)) and of the whole root system (L(pr)) in ECM plants was higher by twofold to fourfold compared with the non-ECM seedlings. HgCl2 had a greater inhibitory effect on L(pc) in ECM compared with non-ECM seedlings, suggesting that the mercury-sensitive, aquaporin (AQP)-mediated water transport was largely responsible for the differences in L(pc) between the two groups of plants. L(pc) was rapidly and drastically reduced by the 50 mM NaCl treatment. However, in ECM plants, the initial decline in L(pc) was followed by a quick recovery to the pre-treatment level, while the reduction of L(pc) in non-ECM seedlings progressed over time. Treatments with fluoride reduced L(pc) by about twofold in non-ECM seedlings and caused smaller reductions of L(pc) in ECM plants. When either 2 mM KF or 2 mM NaF were added to the 50 mM NaCl treatment solution, the inhibitory effect of NaCl on L(pc) was rapidly reversed in both groups of plants. The results suggest that AQP-mediated water transport may be linked to the enhancement of salt stress resistance reported for ECM plants.

Cholesterol depletion induces anoikis-like apoptosis via FAK down-regulation and caveolae internalization.

Caveolae (lipid rafts), microdomains of the plasma membrane, are known to contain various signalling molecules and consequently are involved in the regulation of many biological functions. To investigate the role of the caveolae in cell survival and adhesion, we disrupted the caveolae by depletion of cholesterol, a major lipid component of the caveolae, with methyl-beta cyclodextrin (MbetaCD) treatment of A431 cells. We found that cholesterol depletion induced an anoikis-like cell death involving actin reorganization, resulting in a decrease in cell spreading and an increase in cell detachment, which was reversed by cholesterol addition. Disruption of caveolae led to the down-regulation of FAK, Src activation, tyrosine phosphorylation of caveolin-1 and mobilization of caveolae markers, GM1 and caveolin-1, from the cell surface to the cytoplasm, which were also recovered by cholesterol addition. The expression of dominant-active FAK was able to delay caveolae internalization and apoptosis and attenuated Akt inactivation by MbetaCD, whereas dominant-negative FAK expression resulted in enhanced apoptosis. Moreover, FAK down-regulation by si-RNA resulted in Akt inactivation and thus increased cell death by MbetaCD treatment. Our results suggest that the cholesterol content and/or surface levels of the caveolae affect the activity of FAK, which in turn regulates caveolae internalization and cell survival.

Evolution of nano-grains in high purity copper by accumulative roll-bonding process.

The evolution of nano-grains in oxygen free copper with accumulative roll-bonding is investigated by TEM and EBSD analysis. The ultrafine grains developed in the sample after 8-cycle ARB. It was found that the mean spacing of grain boundaries, which was 63 microm in initial material, reduced to 5.5 microm after 1 cycle, then surprisingly 450 nm after 3 cycles. In addition, the fraction of high-angle boundaries in the sample after 1-cycle ARB was 0.32, but it after 3-cycle ARB was surprisingly more than 0.6. Texture development of the ARB processed samples is different depending on the number of ARB cycles.

Microstructural Changes with Annealing of a Nanostructured Al Alloy Severely Plastic Deformed by Four-Layer Stack Accumulative Roll-Bonding Process.

Microstructural changes with annealing of a nanostructured complex aluminum alloy fabricated by 3 cycles of four-layer stack ARB process using different Al alloys were investigated in detail. The four-layer stack ARB process using AA1050, AA5052 and AA6061 alloy sheets was performed up to 3 cycles without a lubricant at room temperature. The sample fabricated by the ARB is a multi-layer aluminum alloy sheet in which the AA1050, AA5052 and AA6061 aluminum alloys are alternately stacked to each other. The layer thickness of each alloy became thinner and elongated to the rolling direction with the number of ARB cycles. The grain size decreased with increasing of the number of ARB cycles, and became about 160 nm in thickness after 3 cycles. The complex Al alloy still showed ultrafine grained microstructure to annealing temperature of 300 °C, but it had a heterogeneous structure containing both the ultrafine grains and the coarse grains due to an occurrence of discontinuous recrystallization after 350 °C.