Calcium regulation of the Arabidopsis Na+/K+ transporter HKT1;1 improves seed germination under salt stress.

Calcium is known to improve seed-germination rates under salt stress. We investigated the involvement of calcium ions (Ca2+) in regulating HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1; 1), which encodes a Na+/K+ transporter, and its post-translational regulator TYPE 2C PROTEIN PHOSPHATASE 49 (PP2C49), in germinating Arabidopsis (Arabidopsis thaliana) seedlings. Germination rates of hkt1 mutant seeds under salt stress remained unchanged by CaCl2 treatment in wild-type Arabidopsis, whereas pp2c49 mutant seeds displayed improved salt-stress tolerance in the absence of CaCl2 supplementation. Analysis of HKT1;1 and PP2C49 promoter activity revealed that CaCl2 treatment results in radicle-focused expression of HKT1;1 and reduction of the native radicle-exclusive expression of PP2C49. Ion-content analysis indicated that CaCl2 treatment improves K+ retention in germinating wild-type seedlings under salt stress, but not in hkt1 seedlings. Transgenic seedlings designed to exclusively express HKT1;1 in the radicle during germination displayed higher germination rates under salt stress than the wild type in the absence of CaCl2 treatment. Transcriptome analysis of germinating seedlings treated with CaCl2, NaCl, or both revealed 118 upregulated and 94 downregulated genes as responsive to the combined treatment. Bioinformatics analysis of the upstream sequences of CaCl2-NaCl-treatment-responsive upregulated genes revealed the abscisic acid response element CACGTGTC, a potential CaM-binding transcription activator-binding motif, as most prominent. Our findings suggest a key role for Ca2+ in mediating salt-stress responses during germination by regulating genes that function to maintain Na+ and K+ homeostasis, which is vital for seed germination under salt stress.

A Ca2+/CaM-regulated transcriptional switch modulates stomatal development in response to water deficit.

Calcium (Ca2+) signals are decoded by the Ca2+-sensor protein calmodulin (CaM) and are transduced to Ca2+/CaM-binding transcription factors to directly regulate gene expression necessary for acclimation responses in plants. The molecular mechanisms of Ca2+/CaM signal transduction processes and their functional significance remains enigmatic. Here we report a novel Ca2+/CaM signal transduction mechanism that allosterically regulates DNA-binding activity of GT2-LIKE 1 (GTL1), a transrepressor of STOMATAL DENSITY AND DISTRIBUTION 1 (SDD1), to repress stomatal development in response to water stress. We demonstrated that Ca2+/CaM interaction with the 2nd helix of the GTL1 N-terminal trihelix DNA-binding domain (GTL1N) destabilizes a hydrophobic core of GTL1N and allosterically inhibits 3rd helix docking to the SDD1 promoter, leading to osmotic stress-induced Ca2+/CaM-dependent activation (de-repression) of SDD1 expression. This resulted in GTL1-dependent repression of stomatal development in response to water-deficit stress. Together, our results demonstrate that a Ca2+/CaM-regulated transcriptional switch on a trihelix transrepressor directly transduces osmotic stress to repress stomatal development to improve plant water-use efficiency as an acclimation response.

CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6: A Key Regulator of Na+ Homeostasis during Germination.

Salinity impairs seed germination and seedling establishment. We investigated the role of Arabidopsis (Arabidopsis thaliana) CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6 (CAMTA6) in salinity stress responses during early germination. Compared with the wild type, the camta6-4 and camta6-5 mutants were more tolerant to NaCl and abscisic acid (ABA) and accumulated less Na+ In contrast, 4- to 11-d-old camta6 seedlings were more sensitive to NaCl. In camta6, expression of HIGH-AFFINITY K+ TRANSPORTER1 (AtHKT1;1), encoding an Na+/K+ transporter, was restricted to the radicles and was not enhanced by NaCl or ABA. During germination, the camta6 hkt1 double mutant was as sensitive as the wild type and hkt1 to NaCl, suggesting that HKT1;1 is crucial for the salt tolerance of camta6 An ABA response element in the HKT1;1 promoter was found to be indispensable for the enhanced expression of the gene in response to NaCl and to ABA. Transcriptome analysis of the wild type and camta6-5 with and without salt treatment revealed 1,020 up-regulated and 1,467 down-regulated salt-responsive genes in the wild type. Among these, 638 up-regulated and 1,242 down-regulated genes were classified as CAMTA6-dependent. Expression of several known salt stress-associated genes, including SALT OVERLY SENSITIVE1 and Na+/H+ ANTIPORTER, was impaired in camta6 mutants. Bioinformatics analysis of the 5' upstream sequences of the salt-responsive CAMTA6-dependent up-regulated genes revealed the CACGTGTC motif as the most prominent element, representing an ABA response element and a potential CAMTA-binding site. We suggest that CAMTA6 regulates, directly or indirectly, the expression of most of the salt-responsive genes in germinating seeds, including genes that are crucial for Na+ homeostasis and salt stress tolerance.

Transcriptomic analysis of Sorghum bicolor responding to combined heat and drought stress.

BACKGROUND: Abiotic stresses which include drought and heat are amongst the main limiting factors for plant growth and crop productivity. In the field, these stress types are rarely presented individually and plants are often subjected to a combination of stress types. Sorghum bicolor is a cereal crop which is grown in arid and semi-arid regions and is particularly well adapted to the hot and dry conditions in which it originates and is now grown as a crop. In order to better understand the mechanisms underlying combined stress tolerance in this important crop, we have used microarrays to investigate the transcriptional response of Sorghum subjected to heat and drought stresses imposed both individually and in combination. RESULTS: Microarrays consisting of 28585 gene probes identified gene expression changes equating to ~4% and 18% of genes on the chip following drought and heat stresses respectively. In response to combined stress ~20% of probes were differentially expressed. Whilst many of these transcript changes were in common with those changed in response to heat or drought alone, the levels of 2043 specific transcripts (representing 7% of all gene probes) were found to only be changed following the combined stress treatment. Ontological analysis of these 'unique' transcripts identified a potential role for specific transcription factors including MYB78 and ATAF1, chaperones including unique heat shock proteins (HSPs) and metabolic pathways including polyamine biosynthesis in the Sorghum combined stress response. CONCLUSIONS: These results show evidence for both cross-talk and specificity in the Sorghum response to combined heat and drought stress. It is clear that some aspects of the combined stress response are unique compared to those of individual stresses. A functional characterization of the genes and pathways identified here could lead to new targets for the enhancement of plant stress tolerance, which will be particularly important in the face of climate change and the increasing prevalence of these abiotic stress types.

Genome of Acanthamoeba castellanii highlights extensive lateral gene transfer and early evolution of tyrosine kinase signaling.

BACKGROUND: The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. RESULTS: Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. CONCLUSIONS: Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Calcium-regulated transcription in plants.

The past two decades revealed a plethora of Ca2+-responsive proteins and downstream targets in plants, of which several are unique to plants. More recent high-throughput 'omics' approaches and bioinformatics are exposing Ca2+-responsive cis-elements and the corresponding Ca2+-responsive genes. Here, we review the current knowledge on Ca2+-signaling pathways that regulate gene expression in plants, and we link these to mechanisms by which plants respond to biotic and abiotic stresses.

CAMTAs: calmodulin-binding transcription activators from plants to human.

Recently, a novel family of calmodulin-binding transcription activators (CAMTAs) was reported in various eukaryotes. All CAMTAs share a similar domain organization, with a novel type of sequence-specific DNA-binding domain (designated CG-1). This domain could bind DNA directly and activate transcription, or interact with other transcription factors, not through DNA binding, thus acting as a co-activator of transcription. Investigations of CAMTAs in various organisms imply a broad range of functions from sensory mechanisms to embryo development and growth control, highlighted by the apparent involvement of mammalian CAMTA2 in cardiac growth, and of CAMTA1 in tumor suppression and memory performance.

Ca-Responsive cis-Elements in Plants.

External physical and chemical stimuli are transduced via second messengers, following primary interaction with specific membrane or soluble receptors. Ca(2+) is an important second messenger in plants as in other eukaryotes, mediating responses to numerous environmental stimuli and affecting a multitude of cellular processes including gene expression. However, there is yet very little information concerning the cis-elements that mediate Ca(2+)-responsive gene expression. In this article we discuss a recent investigation combining bioinformatics with experimental data, revealing DNA regulatory elements that convey specific cytosolic Ca(2+) transients to the transcription machinery.

Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis.

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.

Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation.

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.