Magnesium Transporter 5 plays an important role in Mg transport for male gametophyte development in Arabidopsis.

During anther development the male gametophyte develops inside the locule and the tapetal cells provide all nutrients for its development. Magnesium Transporter 5 (MGT5) is a member of the MGT family and has dual functions of Mg export and import. Here, we show that male gametophyte mitosis and intine formation are defective in a mgt5 mutant. The transient expression of GFP-MGT5 revealed that MGT5 is localized in the plasma membrane. These findings suggest that in the male gametophyte MGT5 plays a role in importing Mg from the locule and that Mg is essential for male gametophyte development. The expression of MGT5 in the knockout ABORTED MICROSPORES (AMS) mutant (AMS being an essential regulator of tapetum) is tremendously reduced. Chromatin immunoprecipitation and mobility shift assay experiments demonstrated that AMS can directly bind the promoter of MGT5. An immunoelectron microscopy assay revealed that MGT5-His is localized to the plasma membrane of the tapetum. These findings suggest that AMS directly regulates MGT5 in the tapetum and thus induces export of Mg into the locule. The mgt5 plant exhibits severe male sterility while the expression of MGT5 under the tapetum-specific promoter A9 partly rescued mgt5 fertility. mgt5 fertility was restored under high-Mg conditions. These findings suggest that the mgt5 tapetum still has the ability to export Mg and that a sufficient supply of Mg from the tapetum can improve the importation of Mg in the mgt5 male gametophyte. Therefore, MGT5 plays an important role in Mg transport from the tapetum to the microspore.

A rice quantitative trait locus for salt tolerance encodes a sodium transporter.

Many important agronomic traits in crop plants, including stress tolerance, are complex traits controlled by quantitative trait loci (QTLs). Isolation of these QTLs holds great promise to improve world agriculture but is a challenging task. We previously mapped a rice QTL, SKC1, that maintained K(+) homeostasis in the salt-tolerant variety under salt stress, consistent with the earlier finding that K(+) homeostasis is important in salt tolerance. To understand the molecular basis of this QTL, we isolated the SKC1 gene by map-based cloning and found that it encoded a member of HKT-type transporters. SKC1 is preferentially expressed in the parenchyma cells surrounding the xylem vessels. Voltage-clamp analysis showed that SKC1 protein functions as a Na(+)-selective transporter. Physiological analysis suggested that SKC1 is involved in regulating K(+)/Na(+) homeostasis under salt stress, providing a potential tool for improving salt tolerance in crops.

SbHKT1;4, a member of the high-affinity potassium transporter gene family from Sorghum bicolor, functions to maintain optimal Na⁺ /K⁺ balance under Na⁺ stress.

In halophytic plants, the high-affinity potassium transporter HKT gene family can selectively uptake K⁺ in the presence of toxic concentrations of Na⁺. This has so far not been well examined in glycophytic crops. Here, we report the characterization of SbHKT1;4, a member of the HKT gene family from Sorghum bicolor. Upon Na⁺ stress, SbHKT1;4 expression was more strongly upregulated in salt-tolerant sorghum accession, correlating with a better balanced Na⁺ /K⁺ ratio and enhanced plant growth. Heterogeneous expression analyses in mutants of Saccharomyces cerevisiae and Arabidopsis thaliana indicated that overexpressing SbHKT1;4 resulted in hypersensitivity to Na⁺ stress, and such hypersensitivity could be alleviated with the supply of elevated levels of K⁺, implicating that SbHKT1;4 may mediate K⁺ uptake in the presence of excessive Na⁺. Further electrophysiological evidence demonstrated that SbHKT1;4 could transport Na⁺ and K⁺ when expressed in Xenopus laevis oocytes. The relevance of the finding that SbHKT1;4 functions to maintain optimal Na⁺ /K⁺ balance under Na⁺ stress to the breeding of salt-tolerant glycophytic crops is discussed.

The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance.

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.

A rice high-affinity potassium transporter (HKT) conceals a calcium-permeable cation channel.

Plant high-affinity K(+) transport (HKT) proteins are so named because of their relation to bacterial and fungal transporters that mediate high-affinity K(+) uptake. The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held. We have found that one of the rice HKT proteins also functions as a Ca(2+)-permeable cation channel that conducts current carried by a wide range of monovalent and divalent cations. The HKT rice gene, named OsHKT2;4, is expressed in several cell types, including root hairs and vascular parenchyma cells. The protein is localized to the plasma membrane, thereby providing a mechanism for cation uptake and extrusion. This finding goes against firmly entrenched dogma in showing that HKT proteins can function as both ion carriers and channels. The study further extends the function of HKT proteins to Ca(2+)-linked processes and, in so doing, defines a previously undescribed type of Ca(2+)-permeable cation channels in plants. The work also raises questions about the evolutionary changes in this protein family following the divergence of monocots and dicots.

A nitrate transporter encoded by ZmNPF7.9 is essential for maize seed development.

Nitrogen is an essential macronutrient for plants and regulates many aspects of plant growth and development. Nitrate is one of the major forms of nitrogen in plants. However, the role of nitrate uptake and allocation in seed development is not fully understood. Here, we identified the maize (Zea mays) small-kernel mutant zmnpf7.9 and characterized the candidate gene, ZmNPF7.9, which was the same gene as nitrate transport 1.5 (NRT1.5) in maize. This gene is specifically expressed in the basal endosperm transfer layer cells of maize endosperm. Dysfunction of ZmNPF7.9 resulted in delayed endosperm development, abnormal starch deposition and decreased hundred-grain weight. Functional analysis of cRNA-injected Xenopus oocytes showed that ZmNPF7.9 is a low-affinity, pH-dependent bidirectional nitrate transporter. Moreover, the amount of nitrate in mature seeds of the zmnpf7.9 mutant was reduced. These suggest that ZmNPF7.9 is involved in delivering nitrate from maternal tissues to the developing endosperm. Moreover, most of the key genes associated with glycolysis/gluconeogenesis, carbon fixation, carbon metabolism and biosynthesis of amino acids pathways in the zmnpf7.9 mutant were significantly down-regulated. Thus, our results demonstrate that ZmNPF7.9 plays a specific role in seed development and grain weight by regulating nutrition transport and metabolism, which might provide useful information for maize genetic improvement.

AtMGT7: An Arabidopsis gene encoding a low-affinity magnesium transporter.

Magnesium (Mg(2+)) is one of the essential cations in all cells. Although the Mg(2+) transport mechanism has been well-documented in bacteria, less is known about Mg(2+) transporters in eukaryotes. The AtMGT gene family encoding putative magnesium transport proteins had been described previously. We report here that one of the Arabidopsis MGT family members, the AtMGT7 gene, encodes two mRNAs that have resulted from alternative splicing variants, designated AtMGT7a and AtMGT7b. Interestingly, the two mRNA variants were expressed with different patterns with AtMGT7a expressing in all organs, but AtMGT7b appearing only in root and flowers. The AtMGT7a variant functionally complemented a bacterial mutant lacking Mg(2+) transport capacity, whereas AtMGT7b did not. The (63)Ni(2+) tracer uptake analysis in the bacterial model showed that AtMGT7a mediated low-affinity transport of Mg(2+). Consistent with the complementation assay result, (63)Ni(2+) tracer uptake analysis revealed that AtMGT7b did not transport Mg(2+). This study therefore has identified from a higher plant the first low-affinity Mg(2+) transporter encoded by a gene with alternatively spliced transcripts that produce proteins with distinct functions.

16α, 17α-epoxypregnenolone-20-oxime inhibits NO and IL-6 production in LPS-treated RAW264.7 cells.

It has previously been reported that 16α, 17α-epoxypregnenolone-20-oxime (EPREGO) exerts an inhibitory effect on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in microglia. The present study aimed to investigate the effects of EPREGO on the lipopolysaccharide (LPS)­induced inflammatory response in RAW264.7 macrophage cells, and to determine the underlying molecular mechanisms using western blot analysis, enzyme­linked immunosorbent assays and fluorescence­activated cell sorting. The present study demonstrated that LPS­induced production of NO and interleukin (IL)-6, and the protein expression levels of iNOS, were reduced by EPREGO in a dose­ and time­dependent manner, whereas, EPREGO did not affect tumor necrosis factor­α production. In addition, EPREGO suppressed LPS­induced cellular reactive oxygen species production and phagocytosis. Furthermore, EPREGO significantly inhibited the LPS­induced activation of mitogen­activated protein kinases and inhibitor of κB α degradation in LPS­stimulated RAW264.7 cells, thus resulting in modulation of the production of NO and IL­6. Taken together, these results suggest that EPREGO exhibits anti-inflammatory activity in macrophages, thus validating the hypothesis that EPREGO may be useful as a therapeutic agent for the treatment of macrophage-mediated inflammation.

Magnesium transporter AtMGT9 is essential for pollen development in Arabidopsis.

Magnesium (Mg(2+)) is abundant in plant cells and plays a critical role in many physiological processes. A 10-member gene family AtMGT (also known as AtMRS2) was identified in Arabidopsis, which belongs to a eukaryote subset of the CorA superfamily, functioning as Mg(2+) transporters. Some family members (AtMGT1 and AtMGT10) function as high-affinity Mg(2+) transporter and could complement bacterial mutant or yeast mutant lacking Mg(2+) transport capability. Here we report an AtMGT family member, AtMGT9, that functions as a low-affinity Mg(2+) transporter, and is essential for pollen development. The functional complementation assay in Salmonella mutant strain MM281 showed that AtMGT9 is capable of mediating Mg(2+) uptake in the sub-millimolar range of Mg(2+). The AtMGT9 gene was expressed most strongly in mature anthers and was also detectable in vascular tissues of the leaves, and in young roots. Disruption of AtMGT9 gene expression resulted in abortion of half of the mature pollen grains in heterozygous mutant +/mgt9, and no homozygous mutant plant was obtained in the progeny of selfed +/mgt9 plants. Transgenic plants expressing AtMGT9 in these heterozygous plants can recover the pollen phenotype to the wild type. In addition, AtMGT9 RNAi transgenic plants also showed similar abortive pollen phenotype to mutant +/mgt9. Together, our results demonstrate that AtMGT9 functions as a low-affinity Mg(2+) transporter that plays a crucial role in male gametophyte development and male fertility.

Calcineurin-B-like protein CBL9 interacts with target kinase CIPK3 in the regulation of ABA response in seed germination.

Calcium plays a vital role as a second messenger in many signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in calcium signaling by interacting with their interacting protein kinases (CIPKs). In our previous study, we have reported a role for one of the CBLs (CBL9) and one of the CIPKs (CIPK3) in ABA signaling. Here, we have shown that CBL9 and CIPK3 physically and functionally interact with each other in regulating the ABA responses. The CBL9 and CIPK3 proteins interacted with each other in the yeast two-hybrid system and when expressed in plant cells. The double mutant cbl9cipk3 showed the similar hypersensitive response to ABA as observed in single mutants (cbl9 or cipk3). The constitutively active form of CIPK3 genetically complemented the cbl9 mutant, indicating that CIPK3 function downstream of CBL9. Based on these findings, we conclude that CBL9 and CIPK3 act together in the same pathway for regulating ABA responses.

A mitochondrial magnesium transporter functions in Arabidopsis pollen development.

Magnesium is an abundant divalent cation in plant cells and plays a critical role in many physiological processes. We have previously described the identification of a 10-member Arabidopsis gene family encoding putative magnesium transport (MGT) proteins. Here, we report that a member of the MGT family, AtMGT5, functions as a dual-functional Mg-transporter that operates in a concentration-dependent manner, namely it serves as a Mg-importer at micromolar levels and facilitates the efflux in the millimolar range. The AtMGT5 protein is localized in the mitochondria, suggesting that AtMGT5 mediates Mg-trafficking between the cytosol and mitochondria. The AtMGT5 gene was exclusively expressed in anthers at early stages of flower development. Examination of two independent T-DNA insertional mutants of AtMGT5 gene demonstrated that AtMGT5 played an essential role for pollen development and male fertility. This study suggests a critical role for Mg(2+) transport between cytosol and mitochondria in male gametogenesis in plants.

[Isolation, expression analysis of a chilling induced cDNA from rice root with differential display: an evidence role for caffeine-sensitive calcium signal].

Chilling-sensitive rice varieties acquire chilling tolerance when their roots are exposed to water stress for short time. Caffeine-sensitive calcium signal was involved in this procedure. By using total RNA differential display, a chilling induced cDNA(ICT: induction of chilling treatment) was isolated from roots of chilling-sensitive rice variety. It was determined that it is a novel cDNA by homology searching. The transcript level of ict mRNA is up-regulated under chilling stress, it is decreased to low level when the samples were transferred to standard culture conditions. Pre-treated with mannitol for two hours is beneficial to inducing ICT level of expression. This chilling induction was inhibited by caffeine, suggesting that it may play a putative role in signal transduction of caffeine-sensitive calcium.