mRNA cap binding proteins: effects on abscisic acid signal transduction, mRNA processing, and microarray analyses.

The plant hormone abscisic acid (ABA) intricately regulates a multitude of processes during plant growth and development. Recent studies have established a connection between genes participating in various steps of cellular RNA metabolism and the ABA signal transduction machinery. In this chapter we focus on the plant nuclear mRNA cap binding proteins, CBP20 and CBP80. We summarize and report recent findings on their effects on cellular signal transduction networks and mRNA processing events. ABA hypersensitive 1 (abh1) harbors a gene disruption in the Arabidopsis CBP80 gene. Loss-of-function mutation of ABH1 can also result in an early flowering phenotype in the Arabidopsis accession C24. abh1 revealed noncoding cis-natural antisense transcripts (cis-NATs) at the CONSTANS locus in wild-type plants with elevated cis-NAT expression in the mutant. abh1 also revealed an influence on the splicing of the MADS box transcription factor Flowering Locus C pre-mRNA, which may result in the regulation of flowering time. Furthermore, new experiments analyzing complementation of cpb20 with site-directed cpb20 mutants provide evidence that the CAP binding activity of CBP20 is essential for the observed cbp-associated phenotypes. In conclusion, mutants in genes participating in RNA processing provide excellent tools to uncover novel molecular mechanisms for the regulation of RNA metabolism and of signal transduction networks in wild-type plants.

Combining genetics and cell biology to crack the code of plant cell calcium signaling.

Plant hormones, light receptors, pathogens, and abiotic signals trigger elevations in the cytosolic calcium concentration, which mediate physiological and developmental responses. Recent studies are reviewed here that reveal how specific genetic mutations impair or modify stimulus-induced calcium elevations in plant cells. These studies provide genetic evidence for the importance of calcium as a second messenger in plant signal transduction. A fundamental question arises: How can different stimuli use the same second messenger, calcium, to mediate different responses? Recent research and models are reviewed that suggest that several important mechanisms contribute to specificity in calcium signaling in plant cells. These mechanisms include (i) activation of different calcium channels in the plasma membrane and organellar membranes, (ii) stimulus-specific calcium oscillation parameters, (iii) cell type-specific responses, and (iv) intracellular localization of calcium gradients and calcium elevations in plant cells.

An integrated Arabidopsis annotation database for Affymetrix Genechip data analysis, and tools for regulatory motif searches.

Genome-scale sequencing projects have provided the essential information required for the construction of entire genome chips or microarrays for RNA expression studies. The Arabidopsis and rice genomes have been sequenced and whole-genome oligonucleotide arrays are being manufactured. These should soon become available to researchers. Expression studies using genomic-scale expression arrays are providing us with a vast quantity of information at a rapid pace. The rate-limiting step in this type of experiments is not the data generation step but rather the data analysis component of experiments. We report improvements that should facilitate the analysis of Affymetrix Genechip expression data.

Dominant negative guard cell K+ channel mutants reduce inward-rectifying K+ currents and light-induced stomatal opening in arabidopsis.

Inward-rectifying potassium (K+(in)) channels in guard cells have been suggested to provide a pathway for K+ uptake into guard cells during stomatal opening. To test the proposed role of guard cell K+(in) channels in light-induced stomatal opening, transgenic Arabidopsis plants were generated that expressed dominant negative point mutations in the K+(in) channel subunit KAT1. Patch-clamp analyses with transgenic guard cells from independent lines showed that K+(in) current magnitudes were reduced by approximately 75% compared with vector-transformed controls at -180 mV, which resulted in reduction in light-induced stomatal opening by 38% to 45% compared with vector-transformed controls. Analyses of intracellular K+ content using both sodium hexanitrocobaltate (III) and elemental x-ray microanalyses showed that light-induced K+ uptake was also significantly reduced in guard cells of K+(in) channel depressor lines. These findings support the model that K+(in) channels contribute to K+ uptake during light-induced stomatal opening. Furthermore, transpirational water loss from leaves was reduced in the K+(in) channel depressor lines. Comparisons of guard cell K+(in) current magnitudes among four different transgenic lines with different K+(in) current magnitudes show the range of activities of K+(in) channels required for guard cell K+ uptake during light-induced stomatal opening.

Phylogenetic relationships within cation transporter families of Arabidopsis.

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.

An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis.

The plant hormone abscisic acid (ABA) regulates important stress and developmental responses. We have isolated a recessive ABA hypersensitive mutant, abh1, that shows hormone specificity to ABA. ABH1 encodes the Arabidopsis homolog of a nuclear mRNA cap binding protein and functions in a heterodimeric complex to bind the mRNA cap structure. DNA chip analyses show that only a few transcripts are down-regulated in abh1, several of which are implicated in ABA signaling. Consistent with these results, abh1 plants show ABA-hypersensitive stomatal closing and reduced wilting during drought. Interestingly, ABA-hypersensitive cytosolic calcium increases in abh1 guard cells demonstrate amplification of early ABA signaling. Thus, ABH1 represents a modulator of ABA signaling proposed to function by transcript alteration of early ABA signaling elements.

Caenorhabditis elegans expresses a functional phytochelatin synthase.

The formation of phytochelatins, small metal-binding glutathione-derived peptides, is one of the well-studied responses of plants to toxic metal exposure. Phytochelatins have also been detected in some fungi and some marine diatoms. Genes encoding phytochelatin synthases (PCS) have recently been cloned from Arabidopsis, wheat and Schizosaccharomyces pombe. Surprisingly, database searches revealed the presence of a homologous gene in the Caenorhabditis elegans genome, DDBJ/EMBL/GenBank accession no. 266513. Here we show that C. elegans indeed expresses a gene coding for a functional phytochelatin synthase. CePCS complements the Cd2+ sensitivity of a Schizosaccharomyces pombe PCS knock-out strain and confers phytochelatin synthase activity to these cells. Thus, phytochelatins may play a role for metal homeostasis also in certain animals.

A defined range of guard cell calcium oscillation parameters encodes stomatal movements.

Oscillations in cytosolic calcium concentration ([Ca2+]cyt) are central regulators of signal transduction cascades, although the roles of individual [Ca2+]cyt oscillation parameters in regulating downstream physiological responses remain largely unknown. In plants, guard cells integrate environmental and endogenous signals to regulate the aperture of stomatal pores and [Ca2+]cyt oscillations are a fundamental component of stomatal closure. Here we systematically vary [Ca2+]cyt oscillation parameters in Arabidopsis guard cells using a 'calcium clamp' and show that [Ca2+]cyt controls stomatal closure by two mechanisms. Short-term 'calcium-reactive' closure occurred rapidly when [Ca2+]cyt was elevated, whereas the degree of long-term steady-state closure was 'calcium programmed' by [Ca2+]cyt oscillations within a defined range of frequency, transient number, duration and amplitude. Furthermore, in guard cells of the gca2 mutant, [Ca2+]cyt oscillations induced by abscisic acid and extracellular calcium had increased frequencies and reduced transient duration, and steady-state stomatal closure was abolished. Experimentally imposing [Ca2+]cyt oscillations with parameters that elicited closure in the wild type restored long-term closure in gca2 stomata. These data show that a defined window of guard cell [Ca2+]cyt oscillation parameters programs changes in steady-state stomatal aperture.

Guard cell abscisic acid signalling and engineering drought hardiness in plants.

Guard cells are located in the epidermis of plant leaves, and in pairs surround stomatal pores. These control both the influx of CO2 as a raw material for photosynthesis and water loss from plants through transpiration to the atmosphere. Guard cells have become a highly developed system for dissecting early signal transduction mechanisms in plants. In response to drought, plants synthesize the hormone abscisic acid, which triggers closing of stomata, thus reducing water loss. Recently, central regulators of guard cell abscisic acid signalling have been discovered. The molecular understanding of the guard cell signal transduction network opens possibilities for engineering stomatal responses to control CO2 intake and plant water loss.

Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant.

Cytosolic calcium oscillations control signaling in animal cells, whereas in plants their importance remains largely unknown. In wild-type Arabidopsis guard cells abscisic acid, oxidative stress, cold, and external calcium elicited cytosolic calcium oscillations of differing amplitudes and frequencies and induced stomatal closure. In guard cells of the V-ATPase mutant det3, external calcium and oxidative stress elicited prolonged calcium increases, which did not oscillate, and stomatal closure was abolished. Conversely, cold and abscisic acid elicited calcium oscillations in det3, and stomatal closure occurred normally. Moreover, in det3 guard cells, experimentally imposing external calcium-induced oscillations rescued stomatal closure. These data provide genetic evidence that stimulus-specific calcium oscillations are necessary for stomatal closure.

Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells.

Drought is a major threat to agricultural production. Plants synthesize the hormone abscisic acid (ABA) in response to drought, triggering a signalling cascade in guard cells that results in stomatal closure, thus reducing water loss. ABA triggers an increase in cytosolic calcium in guard cells ([Ca2+]cyt) that has been proposed to include Ca2+ influx across the plasma membrane. However, direct recordings of Ca2+ currents have been limited and the upstream activation mechanisms of plasma membrane Ca2+ channels remain unknown. Here we report activation of Ca2+-permeable channels in the plasma membrane of Arabidopsis guard cells by hydrogen peroxide. The H2O2-activated Ca2+ channels mediate both influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard cells. ABA induces the production of H2O2 in guard cells. If H2O2 production is blocked, ABA-induced closure of stomata is inhibited. Moreover, activation of Ca2+ channels by H2O2 and ABA- and H2O2-induced stomatal closing are disrupted in the recessive ABA-insensitive mutant gca2. These data indicate that ABA-induced H2O2 production and the H2O2-activated Ca2+ channels are important mechanisms for ABA-induced stomatal closing.

The Arabidopsis HKT1 gene homolog mediates inward Na(+) currents in xenopus laevis oocytes and Na(+) uptake in Saccharomyces cerevisiae.

The Na(+)-K(+) co-transporter HKT1, first isolated from wheat, mediates high-affinity K(+) uptake. The function of HKT1 in plants, however, remains to be elucidated, and the isolation of HKT1 homologs from Arabidopsis would further studies of the roles of HKT1 genes in plants. We report here the isolation of a cDNA homologous to HKT1 from Arabidopsis (AtHKT1) and the characterization of its mode of ion transport in heterologous systems. The deduced amino acid sequence of AtHKT1 is 41% identical to that of HKT1, and the hydropathy profiles are very similar. AtHKT1 is expressed in roots and, to a lesser extent, in other tissues. Interestingly, we found that the ion transport properties of AtHKT1 are significantly different from the wheat counterpart. As detected by electrophysiological measurements, AtHKT1 functioned as a selective Na(+) uptake transporter in Xenopus laevis oocytes, and the presence of external K(+) did not affect the AtHKT1-mediated ion conductance (unlike that of HKT1). When expressed in Saccharomyces cerevisiae, AtHKT1 inhibited growth of the yeast in a medium containing high levels of Na(+), which correlates to the large inward Na(+) currents found in the oocytes. Furthermore, in contrast to HKT1, AtHKT1 did not complement the growth of yeast cells deficient in K(+) uptake when cultured in K(+)-limiting medium. However, expression of AtHKT1 did rescue Escherichia coli mutants carrying deletions in K(+) transporters. The rescue was associated with a less than 2-fold stimulation of K(+) uptake into K(+)-depleted cells. These data demonstrate that AtHKT1 differs in its transport properties from the wheat HKT1, and that AtHKT1 can mediate Na(+) and, to a small degree, K(+) transport in heterologous expression systems.

Enhancement of Na(+) uptake currents, time-dependent inward-rectifying K(+) channel currents, and K(+) channel transcripts by K(+) starvation in wheat root cells.

Excessive low-affinity Na(+) uptake is toxic to the growth of glycophytic plants. Recently, several reports have suggested that the interaction between K(+) and Na(+) uptake might represent a key factor in determining the Na(+) tolerance of plants. We investigated the effects of K(+) starvation on Na(+) and K(+) uptake mechanisms in the plasma membrane of wheat (Triticum aestivum L.) root cortex cells using the patch-clamp technique. Unexpectedly, K(+) starvation of wheat seedlings was found to enhance the magnitude and frequency of occurrence of time-dependent inward-rectifying K(+) channel currents (I(K)(+)(in)). We examined whether the transcription of a wheat root K(+)(in) channel gene is induced by K(+) starvation. A cDNA coding for a wheat root K(+) channel homolog, TaAKT1 (accession no. AF207745), was isolated. TaAKT1 mRNA levels were up-regulated in roots in response to withdrawal of K(+) from the growth medium. Furthermore, K(+) starvation caused an enhancement of instantaneous Na(+) currents (I(Na)(+)). Electrophysiological analyses suggested that I(K)(+)(in) and I(Na)(+) are not mediated by the same transport protein based on: (a) different activation curves, (b) different time dependencies, (c) different sensitivities to external Ca(2+), and (d) different cation selectivities. These data implicate a role for I(Na)(+) in Na(+) uptake and stress during K(+) starvation, and indicate that K(+)(in) channels may contribute to K(+)-starvation-induced K(+) uptake in wheat roots.

Cadmium and iron transport by members of a plant metal transporter family in Arabidopsis with homology to Nramp genes.

Metal cation homeostasis is essential for plant nutrition and resistance to toxic heavy metals. Many plant metal transporters remain to be identified at the molecular level. In the present study, we have isolated AtNramp cDNAs from Arabidopsis and show that these genes complement the phenotype of a metal uptake deficient yeast strain, smf1. AtNramps show homology to the Nramp gene family in bacteria, yeast, plants, and animals. Expression of AtNramp cDNAs increases Cd(2+) sensitivity and Cd(2+) accumulation in yeast. Furthermore, AtNramp3 and AtNramp4 complement an iron uptake mutant in yeast. This suggests possible roles in iron transport in plants and reveals heterogeneity in the functional properties of Nramp transporters. In Arabidopsis, AtNramps are expressed in both roots and aerial parts under metal replete conditions. Interestingly, AtNramp3 and AtNramp4 are induced by iron starvation. Disruption of the AtNramp3 gene leads to slightly enhanced cadmium resistance of root growth. Furthermore, overexpression of AtNramp3 results in cadmium hypersensitivity of Arabidopsis root growth and increased accumulation of Fe, on Cd(2+) treatment. Our results show that Nramp genes in plants encode metal transporters and that AtNramps transport both the metal nutrient Fe and the toxic metal cadmium.

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells.

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.

Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells.

Elevations in cytoplasmic calcium ([Ca(2)+](cyt)) are an important component of early abscisic acid (ABA) signal transduction. To determine whether defined mutations in ABA signal transduction affect [Ca(2)+](cyt) signaling, the Ca(2)+-sensitive fluorescent dye fura 2 was loaded into the cytoplasm of Arabidopsis guard cells. Oscillations in [Ca(2)+](cyt) could be induced when the external calcium concentration was increased, showing viable Ca(2)+ homeostasis in these dye-loaded cells. ABA-induced [Ca(2)+](cyt) elevations in wild-type stomata were either transient or sustained, with a mean increase of approximately 300 nM. Interestingly, ABA-induced [Ca(2)+](cyt) increases were significantly reduced but not abolished in guard cells of the ABA-insensitive protein phosphatase mutants abi1 and abi2. Plasma membrane slow anion currents were activated in wild-type, abi1, and abi2 guard cell protoplasts by increasing [Ca(2)+](cyt), demonstrating that the impairment in ABA activation of anion currents in the abi1 and abi2 mutants was bypassed by increasing [Ca(2)+](cyt). Furthermore, increases in external calcium alone (which elevate [Ca(2)+](cyt)) resulted in stomatal closing to the same extent in the abi1 and abi2 mutants as in the wild type. Conversely, stomatal opening assays indicated different interactions of abi1 and abi2, with Ca(2)+-dependent signal transduction pathways controlling stomatal closing versus stomatal opening. Together, [Ca(2)+](cyt) recordings, anion current activation, and stomatal closing assays demonstrate that the abi1 and abi2 mutations impair early ABA signaling events in guard cells upstream or close to ABA-induced [Ca(2)+](cyt) elevations. These results further demonstrate that the mutations can be bypassed during anion channel activation and stomatal closing by experimental elevation of [Ca(2)+](cyt).

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast.

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of approximately 55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS-expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS-induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.

Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance.

Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants.