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Salinity is a major problem confronting agriculture in and environments. Sensitivity to high levels of salt in plants is associated with an inability to effectively remove [Na.sup.+] ions from the cell cytoplasm. The ability to compartmentalize [Na.sup.+] may result, in part, from stimulation of the [H.sup.+]-ATPases on the plasma membrane (PM-ATPase) and vacuolar membrane (V-ATPase). These [H.sup.+]-pumping ATPases may provide the driving force for [Na.sup.+] transport via [Na.sup.+]-[H.sup.+] exchangers. In a salt-sensitive line of cotton (Gossypium hirsutum L.), greater relative reductions in root length and root fresh weight than in hypocotyl length of seedlings grown in 75 mM NaCl indicated that the root was most affected by salt stress. To determine if the [H.sup.+]-ATPases are involved in the response to salt, we compared activities of the PMand V-ATPases from roots in salt-sensitive cotton seedlings grown with or without 75 mM NaCl. Higher PM-ATPase activity (42%) was observed in seedlings grown in 75 mM NaCl. This stimulation was specific for [Na.sup.+], was not observed when [Na.sup.+] was added to membrane fractions, and was not due to an increase in PM-ATPase protein levels. V-ATPase protein accumulation was unaffected by growth in the presence of [Na.sup.+], and activity was unaffected by [Na.sup.+] in the growth medium or by [Na.sup.+] added to membrane fractions. These studies suggest that although the PM-ATPase responds to increased [Na.sup.+], activity of the transport proteins on the plasma membrane alone may be insufficient to regulate intracellular [Na.sup.+] levels. In addition, the inability of the V-ATPase to respond to increased levels of [Na.sup.+] indicates that salt sensitivity in cotton seedlings may result, in part, from a lack of effective driving force for compartmentalization of [Na.sup.+]., For Centuries, a major problem facing agriculture in and environments has been the increase in soil salinity, which is typically associated with irrigation (Bernstein and Hayward, 1958). When irrigation water [...]

Abstract Background In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. Results Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. Conclusions We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development.

The presence of varied numbers of CALCINEURIN B-LIKE10 (CBL10) calcium sensor genes in species across the Brassicaceae and the demonstrated role of CBL10 in salt tolerance in Arabidopsis thaliana and Eutrema salsugineum provided a unique opportunity to determine if CBL10 function is modified in different species and linked to salt tolerance. Salinity effects on species growth and cross-species complementation were used to determine the extent of conservation and divergence of CBL10 function in four species representing major lineages within the core Brassicaceae (A. thaliana , E. salsugineum , Schrenkiella parvula , and Sisymbrium irio) as well as the first diverging lineage (Aethionema arabicum). Evolutionary and functional analyses indicate that CBL10 duplicated within expanded lineage II of the Brassicaceae and that, while portions of CBL10 function are conserved across the family, there are species-specific variations in CBL10 function. Paralogous CBL10 genes within a species diverged in expression and function probably contributing to the maintenance of the duplicated gene pairs. Orthologous CBL10 genes diverged in function in a species-specific manner, suggesting that functions arose post-speciation. Multiple CBL10 genes and their functional divergence may have expanded calcium-mediated signaling responses and contributed to the ability of certain members of the Brassicaceae to maintain growth in salt-affected soils. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) plays an essential role in seed germination. In this study, we demonstrate that one SNF1-RELATED PROTEIN KINASE3-type protein kinase, SOS2-LIKE PROTEIN KINASE5 (PKS5), is involved in ABA signal transduction via the phosphorylation of an interacting protein, ABSCISIC ACID-INSENSITIVE5 (ABI5). We found that pks5-3 and pks5-4, two previously identified PKS5 superactive kinase mutants with point mutations in the PKS5 FISL/NAF (a conserved peptide that is necessary for interaction with SOS3 or SOS3-LIKE CALCIUM BINDING PROTEINs) motif and the kinase domain, respectively, are hypersensitive to ABA during seed germination. PKS5 was found to interact with ABI5 in yeast (Saccharomyces cerevisiae), and this interaction was further confirmed in planta using bimolecular fluorescence complementation. Genetic studies revealed that ABI5 is epistatic to PKS5. PKS5 phosphorylates a serine (Ser) residue at position 42 in ABI5 and regulates ABA-responsive gene expression. This phosphorylation was induced by ABA in vivo and transactivated ABI5. Expression of ABI5, in which Ser-42 was mutated to alanine, could not fully rescue the ABA-insensitive phenotypes of the abi5-8 and pks5-4abi5-8 mutants. In contrast, mutating Ser-42 to aspartate rescued the ABA insensitivity of these mutants. These data demonstrate that PKS5-mediated phosphorylation of ABI5 at Ser-42 is critical for the ABA regulation of seed germination and gene expression in Arabidopsis (Arabidopsis thaliana). [ABSTRACT FROM AUTHOR]

Catalases are key regulators of reactive oxygen species homeostasis in plant cells. However, the regulation of catalase activity is not well understood. In this study, we isolated an Arabidopsis thaliana mutant, no catalase activity1-3 (nca1-3) that is hypersensitive to many abiotic stress treatments. The mutated gene was identified by map-based cloning as NCA1, which encodes a protein containing an N-terminal RING-finger domain and a C-terminal tetratricopeptide repeat-like helical domain. NCA1 interacts with and increases catalase activity maximally in a 240-kD complex in planta. In vitro, NCA1 interacts with CATALASE2 (CAT2) in a 1:1 molar ratio, and the NCA1 C terminus is essential for this interaction. CAT2 activity increased 10-fold in the presence of NCA1, and zinc ion binding of the NCA1 N terminus is required for this increase. NCA1 has chaperone protein activity that may maintain the folding of catalase in a functional state. NCA1 is a cytosol-located protein. Expression of NCA1 in the mitochondrion of the nca1-3 mutant does not rescue the abiotic stress phenotypes of the mutant, while expression in the cytosol or peroxisome does. Our results suggest that NCA1 is essential for catalase activity. [ABSTRACT FROM AUTHOR]

The salt stress-induced SALT-OVERLY-SENSHTVE (SOS) pathway in Arabidopsis (Arabidopsis thaliana) involves the perception of a calcium signal by the SOS3 and SOS3-like CALCIUM-BINDING PROTEIN8 (SCaBP8) calcium sensors, which then interact with and activate the SOS2 protein kinase, forming a complex at the plasma membrane that activates the SOS1 Na+/H+ exchanger. It has recently been reported that phosphorylation of SCaBP proteins by SOS2-like protein kinases (PKSs) stabilizes the interaction between the two proteins as part of a regulatory mechanism that was thought to be common to all SCaBP and PKS proteins. Here, we report the calcium-independent activation of PKS24 by SCaBPl and show that activation is dependent on interaction of PKS24 with the C-terminal tail of SCaBPl. However, unlike what has been found for other PKS-SCaBP pairs, multiple amino acids in SCaBPl are phosphorylated by PKS24, and this phosphorylation is dependent on the interaction of the proteins through the PKS24 FISL motif and on the efficient activation of PKS24 by the C-terminal tail of SCaBP1. In addition, we show that Thr-211 and Thr-212, which are not common phosphorylation sites in the conserved PFPF motif found in most SCaBP proteins, are important for this activation. Finally, we also found that SCaBPl-regulated PKS24 kinase activity is important for inactivating the Arabidopsis plasma membrane proton-translocating adenosine triphosphatase. Together, these results suggest the existence of a novel SCaBP-PKS regulatory mechanism in plants. [ABSTRACT FROM AUTHOR]

Microfilament and Ca2+ dynamics play important roles in stress signaling in plants. Through genetic screening of Arabidopsis thaliana mutants that are defective in stress-induced increases in cytosolic Ca2+ ([Ca2+]cyt), we identified Actin-Related Protein2 (Arp2) as a regulator of [Ca2+]cyt in response to salt stress. Plants lacking Arp2 or other proteins in the Arp2/3 complex exhibited enhanced salt-induced increases in [Ca2+]cyt , decreased mitochondria movement, and hypersensitivity to salt. In addition, mitochondria aggregated, the mitochondrial permeability transition pore opened, and mitochondrial membrane potential Ψm was impaired in the arp2 mutant, and these changes were associated with salt-induced cell death. When opening of the enhanced mitochondrial permeability transition pore was blocked or increases in [Ca2+]cyt were prevented, the salt-sensitive phenotype of the arp2 mutant was partially rescued. These results indicate that the Arp2/3 complex regulates mitochondrial-dependent Ca2+ signaling in response to salt stress. [ABSTRACT FROM AUTHOR]

Despite the central importance of noncoding DNA to gene regulation and evolution, understanding of the extent of selection on plant noncoding DNA remains limited compared to that of other organisms. Here we report sequencing of genomes from three Brassicaceae species (Leavenworthia alabamica, Sisymbrium irio and Aethionema arabicum) and their joint analysis with six previously sequenced crucifer genomes. Conservation across orthologous bases suggests that at least 17% of the Arabidopsis thaliana genome is under selection, with nearly one-quarter of the sequence under selection lying outside of coding regions. Much of this sequence can be localized to approximately 90,000 conserved noncoding sequences (CNSs) that show evidence of transcriptional and post-transcriptional regulation. Population genomics analyses of two crucifer species, A. thaliana and Capsella grandiflora, confirm that most of the identified CNSs are evolving under medium to strong purifying selection. Overall, these CNSs highlight both similarities and several key differences between the regulatory DNA of plants and other species. [ABSTRACT FROM AUTHOR]

Halophytes are plants that can naturally tolerate high concentrations of salt in the soil, and their tolerance to salt stress may occur through various evolutionary and molecular mechanisms. Eutrema salsugineum is a halophytic species in the Brassicaceae that can naturally tolerate multiple types of abiotic stresses that typically limit crop productivity, including extreme salinity and cold. It has been widely used as a laboratorial model for stress biology research in plants. Here, we present the reference genome sequence (241Mb) of E. salsugineum at 8x coverage sequenced using the traditional Sanger sequencing-based approach with comparison to its close relative Arabidopsis thaliana. The E. salsugineum genome contains 26,531 protein-coding genes and 51 .4% of its genome is composed of repetitive sequences that mostly reside in pericentromeric regions. Comparative analyses of the genome structures, protein-coding genes, micro RNAs, stress-related pathways, and estimated translation efficiency of proteins between E. salsugineum and A. thaliana suggest that halophyte adaptation to environmental stresses may occur via a global network adjustment of multiple regulatory mechanisms. The E. salsugineum genome provides a resource to identify naturally occurring genetic alterations contributing to the adaptation of halophytic plants to salinity and that might be bioengineered in related crop species. [ABSTRACT FROM AUTHOR]

The plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the regulation of ion and metabolite transport and is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H+-ATPase activity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soil salinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5) negatively regulates the PM H+-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like), activates PM H+-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 are hypersensitive to salt at high external pH and exhibit decreased PM H+-ATPase activity. J3 functions upstream of PKS5 as double mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H+-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our results demonstrate that regulation of PM H+-ATPase activity by J3 takes place via inactivation of the PKS5 kinase. [ABSTRACT FROM AUTHOR]

Background: In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. Results: Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. Conclusions: We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development. [ABSTRACT FROM AUTHOR]

The Salt Overly Sensitive (SOS) pathway plays an important role in the regulation of Na+/K+ ion homeostasis and salt tolerance in Arabidopsis thaliana. Previously, we reported that the calcium binding proteins SOS3 and SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCaBP8) nonredundantly activate the protein kinase SOS2. Here, we show that SOS2 phosphorylates SCaBP8 at its C terminus but does not phosphorylate SOS3. In vitro, SOS2 phosphorylation of SCaBP8 was enhanced by the bimolecular interaction of SOS2 and SCaBP8 and did not require calcium ions. In vivo, this phosphorylation was induced by salt stress, occurred at the membrane, stabilized the SCaBP8-SOS2 interaction, and enhanced plasma membrane Na+/H+ exchange activity. When a Ser at position 237 in the SCaBP8 protein (the SOS2 phosphorylation target) was mutated to Ala, SCaBP8 was no longer phosphorylated by SOS2 and the mutant protein could not fully rescue the salt-sensitive phenotype of the scabp8 mutant. By contrast, when Ser-237 was mutated to Asp to mimic the charge of a phosphorylated Ser residue, the mutant protein rescued the scabp8 salt sensitivity. These data demonstrate that calcium sensor phosphorylation is a critical component of SOS pathway regulation of salt tolerance in Arabidopsis. [ABSTRACT FROM AUTHOR]

Most genes identified that control Arabidopsis trichome initiation and formation are transcription factors or regulatory components in transcriptional networks and include GLABROUS1 (GL1), GLABRA2 (GL2), GLABRA3 (GL3) and TRANSPARENT TESTA GLABRA1 (TTG1). Herein, we report that an importin β-like protein, SENSITIVE TO ABA AND DROUGHT2 (SAD2), is required for trichome initiation. Mutations in SAD2 disrupted trichome initiation resulting in reduced trichome number, but had no effect on trichome development or root hair number and development. Expression levels of GL1, MYB23, GL2 and TTG1 were reduced in shoots of sad2 mutants while expression levels of GL3 and ENHANCER OF GLABRA3 ( EGL3) were enhanced. Overexpression of GL3 increased trichome numbers in wild type but not in sad2 mutants, indicating that the function of the GL3 protein is altered in the sad2 mutants. In contrast, overexpression of GFP-GL1 decreased trichome number in both wild type and sad2. Double mutant analysis of gl1 sad2 and gl3 sad2 indicated that SAD2 functions genetically, at least in part, in the same pathway with these two genes. Co-immunoprecipitation indicated that the sad2 mutation does not disrupt formation of the TTG1-GL3-GL1 complex. Analysis of GFP fusions of GL1, GL2, GL3 and TTG1 suggested that these proteins are most likely not direct cargo of SAD2. Our data suggest that SAD2 is involved in trichome initiation by regulating these nuclear genes. [ABSTRACT FROM AUTHOR]

We report that the Arabidopsis thaliana mutant sensitive to ABA and drought2 (sad2), which harbors a T-DNA insertion in an importin β-like gene, is more tolerant to UV-B radiation than the wild type. Analysis of cyclobutane pyrimidine dimer accumulation revealed that less DNA damage occurred in sad2 than in the wild type during UV-B treatment. No significant growth difference was observed between sad2 and the wild type when treated with the genotoxic drug methyl methanesulfonate, suggesting that SAD2 functions in UV-B protection rather than in DNA damage repair. Whereas the R2R3-type transcription repressor MYB4 has previously been shown to negatively regulate the transcription of cinnamate 4-hydroxylase (C4H) and thus to regulate the synthesis of sinapate esters, expression of both MYB4 and C4H and accumulation of UVabsorbing compounds were significantly higher in sad2 than in the wild type. MYB4 did not localize to the nucleus in the sad2 mutant, suggesting that SAD2 is required for MYB4 nuclear trafficking. SAD2 and MYB4 coimmunoprecipitated, indicating that these proteins localize in the same complex in vivo. MYB4 protein specifically bound to its own promoter in gel shift assays and repressed its own expression, demonstrating that MYB4 protein and mRNA are part of a negative autoregulatory loop. This feedback loop is altered in the sad2 mutant due to the absence of MYB4 protein in the nucleus, leading to the constitutive expression of MYB4 and C4H and resulting in accumulation of UV-absorbing pigments that shield the plant from UV-B radiation. [ABSTRACT FROM AUTHOR]

Regulation of the trans-plasma membrane pH gradient is an important part of plant responses to several hormonal and environmental cues, including auxin, blue light, and fungal elicitors. However, little is known about the signaling components that mediate this regulation. Here, we report that an Arabidopsis thaliana Ser/Thr protein kinase, PKS5, Is a negative regulator of the plasma membrane proton pump (PM H+-ATPase). Loss-of-function pks5 mutant plants are more tolerant of high external pH due to extrusion of protons to the extracellular space. PKS5 phoephorylates the PM H+-ATPase AHA2 at a novel site, Ser-931, in the C-terminal regulatory domain. Phosphorylation at this site inhibits interaction between the PM H+-ATPase and an activating 14-3-3 protein in a yeast expression system. We show that PKS5 interacts with the calcium binding protein SCaBP1 and that high external pH can trigger an increase in the concentration of cytosolic-free calcium. These results suggest that PKS5 is part of a calcium-signaling pathway mediating PM H+-ATPase regulation. [ABSTRACT FROM AUTHOR]