Xingyu Jiang

Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K + /H + Exchanger

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K+ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K+/H+ exchange activity but very little Na+/H+ exchange compared with controls transformed with the empty vector; Na+/H+ exchange was not detected with concentrations of less than 37.5 mM Na+ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K+/H+ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K+ homeostasis.

SpAHA1 and SpSOS1 coordinate in transgenic yeast to improve salt tolerance

In plant cells, the plasma membrane Na+/H+ antiporter SOS1 (salt overly sensitive 1) mediates Na+ extrusion using the proton gradient generated by plasma membrane H+-ATPases, and these two proteins are key plant halotolerance factors. In the present study, two genes from Sesuvium portulacastrum, encoding plasma membrane Na+/H+ antiporter (SpSOS1) and H+-ATPase (SpAHA1), were cloned. Localization of each protein was studied in tobacco cells, and their functions were analyzed in yeast cells. Both SpSOS1 and SpAHA1 are plasma membrane-bound proteins. Real-time polymerase chain reaction (PCR) analyses showed that SpSOS1 and SpAHA1 were induced by salinity, and their expression patterns in roots under salinity were similar. Compared with untransformed yeast cells, SpSOS1 increased the salt tolerance of transgenic yeast by decreasing the Na+ content. The Na+/H+ exchange activity at plasma membrane vesicles was higher in SpSOS1-transgenic yeast than in the untransformed strain. No change was observed in the salt tolerance of yeast cells expressing SpAHA1 alone; however, in yeast transformed with both SpSOS1 and SpAHA1, SpAHA1 generated an increased proton gradient that stimulated the Na+/H+ exchange activity of SpSOS1. In this scenario, more Na+ ions were transported out of cells, and the yeast cells co-expressing SpSOS1 and SpAHA1 grew better than the cells transformed with only SpSOS1 or SpAHA1. These findings demonstrate that the plasma membrane Na+/H+ antiporter SpSOS1 and H+-ATPase SpAHA1 can function in coordination. These results provide a reference for developing more salt-tolerant crops via co-transformation with the plasma membrane Na+/H+ antiporter and H+-ATPase.

Characterization of Ca2+/H+ exchange in the plasma membrane of Saccharomyces cerevisiae

The characteristics of the Ca(2+)/H(+) exchange were directly investigated in functionally inverted (inside-out) plasma membrane vesicles isolated from yeast using an aqueous two-phase partitioning method. Results showed that following the generation of an inside-acid pH gradient (fluorescence quenching), addition of Ca(2+) caused movement of H(+) out of the vesicles (fluorescence recovery). The Ca(2+)/H(+) exchange displayed saturation kinetics with respect to extravesicular Ca(2+) and ATP concentrations in the plasma membrane, and showed specificity for Ca(2+). The protonophore FCCP (carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone), abolished the fluorescence quenching and consequently inhibited Ca(2+)/H(+) exchange in plasma membrane vesicles. Vanadate, which is known to inhibit the plasma membrane H(+)-ATPase, significantly decreased the Ca(2+)-dependent transport of H(+) out of vesicles. When the electrical potential across the plasma membrane was dissipated with valinomycin and potassium, the rate of Ca(2+)/H(+) exchange increased compared to that of the control without valinomycin, indicating that the stoichiometric ratio for this exchange is greater than 2H(+):Ca(2+). These data suggest that Ca(2+) is transported out of yeast cells through a Ca(2+)/H(+) exchange system that is driven by the proton-motive force generated by the plasma membrane H(+)-ATPase.

Hyperactive mutant of a wheat plasma membrane Na+/H+ antiporter improves the growth and salt tolerance of transgenic tobacco

Wheat SOS1 (TaSOS1) activity could be relieved upon deletion of the C-terminal 168 residues (the auto-inhibitory domain). This truncated form of wheat SOS1 (TaSOS1-974) was shown to increase compensation (compared to wild-type TaSOS1) for the salt sensitivity of a yeast mutant strain, AXT3K, via increased Na+ transportation out of cells during salinity stress. Expression of the plasma membrane proteins TaSOS1-974 or TaSOS1 improved the growth of transgenic tobacco plants compared with wild-type plants under normal conditions. However, plants expressing TaSOS1-974 grew better than TaSOS1-transformed plants. Upon salinity stress, Na+ efflux and K+ influx rates in the roots of transgenic plants expressing TaSOS1-974 or TaSOS1 were greater than those of wild-type plants. Furthermore, compared to TaSOS1-transgenic plants, TaSOS1-974-expressing roots showed faster Na+ efflux and K+ influx, resulting in less Na+ and more K+ accumulation in TaSOS1-974-transgenic plants compared to TaSOS1-transgenic and wild-type plants. TaSOS1-974-expressing plants had the lowest MDA content and electrolyte leakage among all tested plants, indicating that TaSOS1-974 might protect the plasma membrane against oxidative damage generated by salt stress. Overall, TaSOS1-974 conferred higher salt tolerance in transgenic plants compared to TaSOS1. Consistent with this result, transgenic plants expressing TaSOS1-974 showed a better growth performance than TaSOS1-expressing and wild-type plants under saline conditions.

Boron deficiency affects cell morphology and structure of young leaves of radish

Boron (B) is an essential microelement for the growth and development of plants. B-deficient radish plants grew slowly compared to B-sufficient controls. Soluble B and cell wall-bound B decreased in young leaves on removal of B from culture medium. In old leaves, B deficiency reduced soluble B content but there was no significant effect on cell wall-bound B content compared to controls. The mesophyll cells in the middle of leaves were enlarged abnormally and had greater cell wall thickness under B-deficient conditions. B deficiency reduced the stomata frequency, inhibited the stomata aperture, and guard cells had thickened cell walls. B-starved leaves showed decreased photosynthesis and stomatal conductance. These indicate that B deficiency could interfere with cell wall development, especially irregular guard cell walls as a result of B deficiency severely affected the rhythmic stomatal closing and opening, preventing the normal functioning of stomata. Correspondingly, photosynthesis was indirectly affected, and plant growth decreased.

The response of Mo-hydroxylases and abscisic acid to salinity in wheat genotypes with differing salt tolerances

The differential responses of the wheat cultivars Shi4185 and Yumai47 to salinity were studied. The higher sensitivity of Yumai47 to salinity was linked to a greater growth reduction under salt stress, compared to more salt-tolerant Shi4185. Salinity increased the Na+, proline and superoxide anion radical (O2−) contents in both cultivars. Leaf Na+ content increased less in the more salt-tolerant cultivar Shi4185 than salt-sensitive Yumai47. The proline content increased more significantly in Shi4185 than Yumai47; on the contrary, superoxide anion radical content increased less in Shi4185 than Yumai47. This data indicated that wheat salinity tolerance can be increased by controlling Na+ transport from the root to shoot, associated with higher osmotic adjustment capability and antioxidant activity. Although salinity increased aldehyde oxidase (AO) activity and abscisic acid (ABA) content in the leaves and roots of both cultivars following the addition of NaCl to the growth medium, AO and ABA increased more in the salt-sensitive cultivar Yumai47 than the more salt-tolerant cultivar Shi4185. Xanthine dehydrogenase (XDH) activity in the leaves of both cultivars increased with increasing concentrations of NaCl; however, leaf XDH activity increased more significantly in Yumai47 than Shi4185. Root XDH activity in Shi4185 decreased with increasing NaCl concentrations, whereas salinity induced an increased root XDH activity in Yumai47. The involvement of AO and XDH enzymatic activities and altered ABA content in the response mechanisms of wheat to salinity are discussed herein.

Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL) and 26 CIPK (MeCIPK) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

Over-expression of a plasma membrane H + -ATPase SpAHA1 conferred salt tolerance to transgenic Arabidopsis

The SpAHA1 gene, encoding a plasma membrane (PM) H+-ATPase (AHA) in Sesuvium portulacastrum, was transformed into Arabidopsis plants, and its expression increased salinity tolerance of transgenic Arabidopsis plants: seed germination ratio, root growth, and biomass of transgenic plants were greater compared to wild-type plants under NaCl treatment condition. Upon salinity stress, both Na+ and H+ effluxes in the roots of SpAHA1 expressing plants were faster than those of untransformed plants. Transformed plants with SpAHA1 had lower Na+ and higher K+ contents relative to wild-type plants when treated with NaCl, resulting in greater K+/Na+ ratio in transgenic plants than in wild-type plants under salt stress. Extent of oxidative stress increased in both transgenic and wild-type plants exposed to salinity stress, but overexpression of SpAHA1 could alleviate the accumulation of hydrogen peroxide (H2O2) induced by NaCl treatment in transgenic plants relative to wild-type plants; the content of malondialdehyde (MDA) was lower in transgenic plants than that in wild-type plants under salinity stress. These results suggest that the higher H+-pumping activity generated by SpAHA1 improved the growth of transgenic plants via regulating ion and reactive oxygen species (ROS) homeostasis in plant cells under salinity stress.

The Sesuvium portulacastrum Plasma Membrane Na+/H+ Antiporter SpSOS1 Complemented the Salt Sensitivity of Transgenic Arabidopsis sos1 Mutant Plants

The plasma membrane (PM) Na+/H+ antiporter SOS1 (salt overly sensitive 1) has emerged as a key factor in regulating plant salt tolerance. The SpSOS1 gene, which encodes a PM Na+/H+ antiporter, was cloned from the halophyte Sesuvium portulacastrum and transformed into Arabidopsis sos1 mutant plants. As shown from the results, the SpSOS1 expression complemented the salt sensitivity of the sos1 mutant plants. Upon salinity stress, SpSOS1-transgenic Arabidopsis sos1 mutant seeds displayed higher germination ratio compared to the sos1 mutant. The sos1 mutant plants expressing SpSOS1 grew better and had a lower Na+/K+ ratio than that of the sos1 mutant and wild-type (WT) plants when they were treated with NaCl. In addition, SpSOS1-overexpressed Arabidopsis accumulated less malondialdehyde (MDA) and had a lower level of electrolyte leakage than that in the sos1 mutant and WT plants under salt stress. Furthermore, the SpSOS1 expression in transgenic sos1 mutant plants also increased the transcript levels of some salt stress-related genes, such as AtHKT1;1 (high-affinity K+ transporter 1;1), AtSOS2 (salt overly sensitive 2), AtSCABP8 (SOS3-like calcium binding protein 8), and AtNHX1 (Na+/H+ exchanger 1). These results suggested that SpSOS1 improved the plant salt tolerance by regulating ion homeostasis and protecting the plasma membrane against oxidative damage under salt stress.