PLANT PHYSIOLOGY ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

A Nuclear Factor Regulates Abscisic Acid Responses in Arabidopsis

Kim, M. J., Shin, R., Schachtman, D. P. — Tue, 03 Nov 2009 12:22:27 PST

Abscisic acid (ABA) is a plant hormone that regulates plant growth as well as stress responses. In this study, we identified and characterized a new Arabidopsis (Arabidopsis thaliana) protein, Nuclear Protein X1 (NPX1), which was up-regulated by stress and treatment with exogenous ABA. Stomatal closure, seed germination, and primary root growth are well-known ABA responses that were less sensitive to ABA in NPX1-overexpressing plants. NPX1-overexpressing plants were more drought sensitive, and the changes in response to drought were due to the altered guard cell sensitivity to ABA in transgenic plants and not to a lack of ABA production. The nuclear localization of NPX1 correlated with changes in the expression of genes involved in ABA biosynthesis and ABA signal transduction. To understand the function of NPX1, we searched for interacting proteins and found that an ABA-inducible NAC transcription factor, TIP, interacted with NPX1. Based on the whole plant phenotypes, we hypothesized that NPX1 acts as a transcriptional repressor, and this was demonstrated in yeast, where we showed that TIP was repressed by NPX1. Our results indicate that the previously unknown protein NPX1 acts as a negative regulator in plant response to changes in environmental conditions through the control of ABA-regulated gene expression. The characterization of this factor enhances our understanding of guard cell function and the mechanisms that plants use to modulate water loss from leaves under drought conditions.

Hormone- and Light-Mediated Regulation of Heat-Induced Differential Petiole Growth in Arabidopsis

van Zanten, M., Voesenek, L. A.C.J., Peeters, A. J.M., Millenaar, F. F. — Tue, 03 Nov 2009 12:22:27 PST

Plants react quickly and profoundly to changes in their environment. A sudden increase in temperature, for example, induces differential petiole growth-driven upward leaf movement (hyponastic growth) in Arabidopsis (Arabidopsis thaliana). We show that accessions that face the strongest fluctuations in diurnal temperature in their natural habitat are least sensitive for heat-induced hyponastic growth. This indicates that heat-induced hyponastic growth is a trait subject to natural selection. The response is induced with kinetics remarkably similar to ethylene- and low light-induced hyponasty in several accessions. Using pharmacological assays, transcript analysis, and mutant analyses, we demonstrate that ethylene and the photoreceptor protein phytochrome B are negative regulators of heat-induced hyponastic growth and that low light, phytochrome A, auxin, polar auxin transport, and abscisic acid are positive regulators of heat-induced hyponastic growth. Furthermore, auxin, auxin polar transport, phytochrome A, phytochrome B, and cryptochromes are required for a fast induction of heat-induced hyponastic growth.

Phosphate (Pi) Starvation Effect on the Cytosolic Pi Concentration and Pi Exchanges across the Tonoplast in Plant Cells: An in Vivo 31P-Nuclear Magnetic Resonance Study Using Methylphosphonate as a Pi Analog

Pratt, J., Boisson, A.-M., Gout, E., Bligny, R., Douce, R., Aubert, S. — Tue, 03 Nov 2009 12:22:28 PST

In vivo ³¹P-NMR analyses showed that the phosphate (Pi) concentration in the cytosol of sycamore (Acer pseudoplatanus) and Arabidopsis (Arabidopsis thaliana) cells was much lower than the cytoplasmic Pi concentrations usually considered (60–80 µm instead of >1 mm) and that it dropped very rapidly following the onset of Pi starvation. The Pi efflux from the vacuole was insufficient to compensate for the absence of external Pi supply, suggesting that the drop of cytosolic Pi might be the first endogenous signal triggering the Pi starvation rescue metabolism. Successive short sequences of Pi supply and deprivation showed that added Pi transiently accumulated in the cytosol, then in the stroma and matrix of organelles bounded by two membranes (plastids and mitochondria, respectively), and subsequently in the vacuole. The Pi analog methylphosphonate (MeP) was used to analyze Pi exchanges across the tonoplast. MeP incorporated into cells via the Pi carrier of the plasma membrane; it accumulated massively in the cytosol and prevented Pi efflux from the vacuole. This blocking of vacuolar Pi efflux was confirmed by in vitro assays with purified vacuoles. Subsequent incorporation of Pi into the cells triggered a massive transfer of MeP from the cytosol to the vacuole. Mechanisms for Pi exchanges across the tonoplast are discussed in the light of the low cytosolic Pi level, the cell response to Pi starvation, and the Pi/MeP interactive effects.

Complexation and Toxicity of Copper in Higher Plants. I. Characterization of Copper Accumulation, Speciation, and Toxicity in Crassula helmsii as a New Copper Accumulator

Kupper, H., Gotz, B., Mijovilovich, A., Kupper, F. C., Meyer-Klaucke, W. — Thu, 01 Oct 2009 06:20:40 PDT

The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 µm (=0.6 ppm) Cu²⁺ in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, ¹³C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.

Complexation and Toxicity of Copper in Higher Plants. II. Different Mechanisms for Copper versus Cadmium Detoxification in the Copper-Sensitive Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype)

Thu, 01 Oct 2009 06:20:41 PDT

The cadmium/zinc hyperaccumulator Thlaspi caerulescens is sensitive toward copper (Cu) toxicity, which is a problem for phytoremediation of soils with mixed contamination. Cu levels in T. caerulescens grown with 10 µm Cu²⁺ remained in the nonaccumulator range (<50 ppm), and most individuals were as sensitive toward Cu as the related nonaccumulator Thlaspi fendleri. Obviously, hyperaccumulation and metal resistance are highly metal specific. Cu-induced inhibition of photosynthesis followed the "sun reaction" type of damage, with inhibition of the photosystem II reaction center charge separation and the water-splitting complex. A few individuals of T. caerulescens were more Cu resistant. Compared with Cu-sensitive individuals, they recovered faster from inhibition, at least partially by enhanced repair of chlorophyll-protein complexes but not by exclusion, since the content of Cu in their shoots was increased by about 25%. Extended x-ray absorption fine structure (EXAFS) measurements on frozen-hydrated leaf samples revealed that a large proportion of Cu in T. caerulescens is bound by sulfur ligands. This is in contrast to the known binding environment of cadmium and zinc in the same species, which is dominated by oxygen ligands. Clearly, hyperaccumulators detoxify hyperaccumulated metals differently compared with nonaccumulated metals. Furthermore, strong features in the Cu-EXAFS spectra ascribed to metal-metal contributions were found, in particular in the Cu-resistant specimens. Some of these features may be due to Cu binding to metallothioneins, but a larger proportion seems to result from biomineralization, most likely Cu(II) oxalate and Cu(II) oxides. Additional contributions in the EXAFS spectra indicate complexation of Cu(II) by the nonproteogenic amino acid nicotianamine, which has a very high affinity for Cu(II) as further characterized here.

Heterotrimeric G Protein Signaling Is Required for Epidermal Cell Death in Rice

Steffens, B., Sauter, M. — Thu, 01 Oct 2009 06:20:41 PDT

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H₂O₂). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP--S induced epidermal cell death, whereas GDP-β-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the G subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H₂O₂ was nearly completely abolished, indicating that signaling through G is essential. Ethylene and H₂O₂ were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of G. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that G activity is regulated posttranscriptionally.

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase, AtNUDX7, Is Involved in the Response to Oxidative Stress

Thu, 01 Oct 2009 06:20:41 PDT

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADP-ribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro_35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro_35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro_35S:AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro_35S:AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD⁺ and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro_35S:AtNUDX7 plants. Accumulation of NAD⁺ and ATP was observed in the KO-nudx7- and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD⁺ by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.

Nitric Reductase-Dependent Nitric Oxide Production Is Involved in Cold Acclimation and Freezing Tolerance in Arabidopsis

Zhao, M.-G., Chen, L., Zhang, L.-L., Zhang, W.-H. — Thu, 01 Oct 2009 06:20:41 PDT

Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. We evaluated the role of NO in cold acclimation and freezing tolerance using Arabidopsis (Arabidopsis thaliana) wild type and mutants nia1nia2 (for nitrate reductase [NR]-defective double mutant) and Atnoa1/rif1 (for nitric oxide associated1/resistant to inhibition by fosmidomycin1) that exhibit defects in NR and reduced NO production, respectively. Cold acclimation induced an increase in endogenous NO production in wild-type and Atnoa1/rif1 leaves, while endogenous NO level in nia1nia2 leaves was lower than in wild-type ones and was little changed during cold acclimation. Cold acclimation stimulated NR activity and induced up-regulation of NIA1 gene expression. In contrast, cold acclimation reduced the quantity of NOA1/RIF1 protein and inhibited NO synthase (NOS) activity. These results indicate that up-regulation of NR-dependent NO synthesis underpins cold acclimation-induced NO production. Seedlings of nia1nia2 were less tolerant to freezing than wild-type plants. Pharmacological studies using NR inhibitor, NO scavenger, and NO donor showed that NR-dependent NO level was positively correlated with freezing tolerance. Furthermore, cold acclimation up- and down-regulated expression of P5CS1 and ProDH genes, respectively, resulting in enhanced accumulation of proline (Pro) in wild-type plants. The stimulation of Pro accumulation by cold acclimation was reduced by NR inhibitor and NO scavenger, while Pro accumulation by cold acclimation was not affected by the NOS inhibitor. In contrast to wild-type plants, cold acclimation up-regulated ProDH gene expression in nia1nia2 plants, leading to less accumulation in nia1nia2 plants than in wild-type plants. These findings demonstrate that NR-dependent NO production plays an important role in cold acclimation-induced increase in freezing tolerance by modulating Pro accumulation in Arabidopsis.

Arabidopsis Putative Selenium-Binding Protein1 Expression Is Tightly Linked to Cellular Sulfur Demand and Can Reduce Sensitivity to Stresses Requiring Glutathione for Tolerance

Thu, 01 Oct 2009 06:20:41 PDT

Selenium-Binding Protein1 (SBP1) gene expression was studied in Arabidopsis (Arabidopsis thaliana) seedlings challenged with several stresses, including cadmium (Cd), selenium {selenate [Se(VI)] and selenite [Se(IV)]}, copper (Cu), zinc (Zn), and hydrogen peroxide (H₂O₂) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1::LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H₂O₂ but not in response to Se(IV) or Zn. The pattern of expression of SBP1 was similar to that of PRH43, which encodes the 5'-Adenylylphosphosulfate Reductase2, a marker for the induction of the sulfur assimilation pathway, suggesting that an enhanced sulfur demand triggers SBP1 up-regulation. Correlated to these results, SBP1 promoter showed enhanced activity in response to sulfur starvation. The sulfur starvation induction of SBP1 was abolished by feeding the plants with glutathione (GSH) and was enhanced when seedlings were treated simultaneously with buthionine sulfoxide, which inhibits GSH synthesis, indicating that GSH level participates in the regulation of SBP1 expression. Changes in total GSH level were observed in seedlings challenged with Cd, Se(VI), and H₂O₂. Accordingly, cad2-1 seedlings, affected in GSH synthesis, were more sensitive than wild-type plants to these three stresses. Moreover, wild-type and cad2-1 seedlings overexpressing SBP1 showed a significant enhanced tolerance to Se(VI) and H₂O₂ in addition to the previously described resistance to Cd, highlighting that SBP1 expression decreases sensitivity to stress requiring GSH for tolerance. These results are discussed with regard to the potential regulation and function of SBP1 in plants.

Expression of Pyrococcus furiosus Superoxide Reductase in Arabidopsis Enhances Heat Tolerance

Im, Y. J., Ji, M., Lee, A., Killens, R., Grunden, A. M., Boss, W. F. — Thu, 01 Oct 2009 06:20:41 PDT

Plants produce reactive oxygen species (ROS) in response to environmental stresses sending signaling cues, which, if uncontrolled, result in cell death. Like other aerobic organisms, plants have ROS-scavenging enzymes, such as superoxide dismutase (SOD), which removes superoxide anion radical (O₂^–) and prevents the production and buildup of toxic free radicals. However, increasing the expression of cytosolic SODs is complex, and increasing their production in vivo has proven to be challenging. To avoid problems with endogenous regulation of gene expression, we expressed a gene from the archaeal hyperthermophile Pyrococcus furiosus that reduces O₂^–. P. furiosus uses superoxide reductase (SOR) rather than SOD to remove superoxide. SOR is a thermostable enzyme that reduces O₂^– in a one-electron reduction without producing oxygen. We show that P. furiosus SOR can be produced as a functional enzyme in planta and that plants producing SOR have enhanced tolerance to heat, light, and chemically induced ROS. Stress tolerance in the SOR-producing plants correlates positively with a delayed increase in ROS-sensitive transcripts and a decrease in ascorbate peroxidase activity. The SOR plants provide a good model system to study the impact of cytosolic ROS on downstream signaling in plant growth and development. Furthermore, this work demonstrates that this synthetic approach for reducing cytosolic ROS holds promise as a means for improving stress tolerance in crop plants.

Abnormal Physiological and Molecular Mutant Phenotypes Link Chloroplast Polynucleotide Phosphorylase to the Phosphorus Deprivation Response in Arabidopsis

Thu, 01 Oct 2009 06:20:41 PDT

A prominent enzyme in organellar RNA metabolism is the exoribonuclease polynucleotide phosphorylase (PNPase), whose reversible activity is governed by the nucleotide diphosphate-inorganic phosphate ratio. In Chlamydomonas reinhardtii, PNPase regulates chloroplast transcript accumulation in response to phosphorus (P) starvation, and PNPase expression is repressed by the response regulator PSR1 (for PHOSPHORUS STARVATION RESPONSE1) under these conditions. Here, we investigated the role of PNPase in the Arabidopsis (Arabidopsis thaliana) P deprivation response by comparing wild-type and pnp mutant plants with respect to their morphology, metabolite profiles, and transcriptomes. We found that P-deprived pnp mutants develop aborted clusters of lateral roots, which are characterized by decreased auxin responsiveness and cell division, and exhibit cell death at the root tips. Electron microscopy revealed that the collapse of root organelles is enhanced in the pnp mutant under P deprivation and occurred with low frequency under P-replete conditions. Global analyses of metabolites and transcripts were carried out to understand the molecular bases of these altered P deprivation responses. We found that the pnp mutant expresses some elements of the deprivation response even when grown on a full nutrient medium, including altered transcript accumulation, although its total and inorganic P contents are not reduced. The pnp mutation also confers P status-independent responses, including but not limited to stress responses. Taken together, our data support the hypothesis that the activity of the chloroplast PNPase is involved in plant acclimation to P availability and that it may help maintain an appropriate balance of P metabolites even under normal growth conditions.

Loss of Halophytism by Interference with SOS1 Expression

Wed, 02 Sep 2009 10:00:32 PDT

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na⁺) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na⁺ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na⁺ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na⁺ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na⁺ as Arabidopsis.

Strain Mechanosensing Quantitatively Controls Diameter Growth and PtaZFP2 Gene Expression in Poplar

Wed, 02 Sep 2009 10:00:32 PDT

Mechanical signals are important factors that control plant growth and development. External mechanical loadings lead to a decrease in elongation and a stimulation of diameter growth, a syndrome known as thigmomorphogenesis. A previous study has demonstrated that plants perceive the strains they are subjected to and not forces or stresses. On this basis, an integrative biomechanical model of mechanosensing was established ("sum-of-strains model") and tested on tomato (Solanum lycopersicum) elongation but not for local responses such as diameter growth or gene expression. The first aim of this interdisciplinary work was to provide a quantitative study of the effect of a single transitory bending on poplar (Populus tremula x alba) diameter growth and on the expression level of a primary mechanosensitive transcription factor gene, PtaZFP2. The second aim of this work was to assess the sum-of-strains model of mechanosensing on these local responses. An original bending device was built to study stem responses according to a controlled range of strains. A single bending modified plant diameter growth and increased the relative abundance of PtaZFP2 transcripts. Integrals of longitudinal strains induced by bending on the responding tissues were highly correlated to local plant responses. The sum-of-strains model of mechanosensing established for stem elongation was thus applicable for local responses at two scales: diameter growth and gene expression. These novel results open avenues for the ordering of gene expression profiles as a function of the intensity of mechanical stimulation and provide a generic biomechanical core for an integrative model of thigmomorphogenesis linking gene expression with growth responses.

Overexpressing AtPAP15 Enhances Phosphorus Efficiency in Soybean

Wang, X., Wang, Y., Tian, J., Lim, B. L., Yan, X., Liao, H. — Wed, 02 Sep 2009 10:00:32 PDT

Low phosphorus (P) availability is a major constraint to crop growth and production, including soybean (Glycine max), on a global scale. However, 50% to 80% of the total P in agricultural soils exists as organic phosphate, which is unavailable to plants unless hydrolyzed to release inorganic phosphate. One strategy for improving crop P nutrition is the enhanced activity of acid phosphatases (APases) to obtain or remobilize inorganic phosphate from organic P sources. In this study, we overexpressed an Arabidopsis (Arabidopsis thaliana) purple APase gene (AtPAP15) containing a carrot (Daucus carota) extracellular targeting peptide in soybean hairy roots and found that the APase activity was increased by 1.5-fold in transgenic hairy roots. We subsequently transformed soybean plants with AtPAP15 and studied three homozygous overexpression lines of AtPAP15. The three transgenic lines exhibited significantly improved P efficiency with 117.8%, 56.5%, and 57.8% increases in plant dry weight, and 90.1%, 18.2%, and 62.6% increases in plant P content, respectively, as compared with wild-type plants grown on sand culture containing phytate as the sole P source. The transgenic soybean lines also exhibited a significant level of APase and phytase activity in leaves and root exudates, respectively. Furthermore, the transgenic lines exhibited improved yields when grown on acid soils, with 35.9%, 41.0%, and 59.0% increases in pod number per plant, and 46.0%, 48.3%, and 66.7% increases in seed number per plant. Taken together, to our knowledge, our study is the first report on the improvement of P efficiency in soybean through constitutive expression of a plant APase gene. These findings could have significant implications for improving crop yield on soils low in available P, which is a serious agricultural limitation worldwide.

BOBBER1 Is a Noncanonical Arabidopsis Small Heat Shock Protein Required for Both Development and Thermotolerance

Wed, 02 Sep 2009 10:00:32 PDT

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here, we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis (Arabidopsis thaliana) small heat shock protein. BOB1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss-of-function mutants are embryo lethal, we used a partial loss-of-function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and postembryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants, BOB1 is cytoplasmic at basal temperatures, but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects, bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide, to our knowledge, the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis

Wed, 02 Sep 2009 10:00:32 PDT

The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.

Physiological and Transcriptome Analysis of Iron and Phosphorus Interaction in Rice Seedlings

Wed, 02 Sep 2009 10:00:32 PDT

The antagonistic interaction between iron (Fe) and phosphorus (P) has been noted in the area of plant nutrition. To understand the physiology and molecular mechanisms of this interaction, we studied the growth performance, nutrient concentration, and gene expression profiles of root and shoot segments derived from 10-d-old rice (Oryza sativa) seedlings under four different nutrient conditions: (1) full strength of Fe and P (+Fe+P); (2) full strength of P and no Fe (–Fe+P); (3) full strength of Fe and no P (+Fe–P); and (4) without both Fe and P (–Fe–P). While removal of Fe in the growth medium resulted in very low shoot and root Fe concentrations, the chlorotic symptoms and retarded seedling growth were only observed on seedlings grown in the presence of P. Microarray data showed that in roots, 7,628 transcripts were significantly changed in abundance in the absence of Fe alone. Interestingly, many of these changes were reversed if P was also absent (–Fe–P), with only approximately 15% overlapping with –Fe alone (–Fe+P). Analysis of the soluble Fe concentration in rice seedling shoots showed that P deficiency resulted in significantly increased Fe availability within the plants. The soluble Fe concentration under –Fe–P conditions was similar to that under +Fe+P conditions. These results provide evidence that the presence of P can affect Fe availability and in turn can influence the regulation of Fe-responsive genes.

The MYB96 Transcription Factor Mediates Abscisic Acid Signaling during Drought Stress Response in Arabidopsis

Wed, 02 Sep 2009 10:00:32 PDT

Plant adaptive responses to drought are coordinated by adjusting growth and developmental processes as well as molecular and cellular activities. The root system is the primary site that perceives drought stress signals, and its development is profoundly affected by soil water content. Various growth hormones, particularly abscisic acid (ABA) and auxin, play a critical role in root growth under drought through complex signaling networks. Here, we report that a R2R3-type MYB transcription factor, MYB96, regulates drought stress response by integrating ABA and auxin signals. The MYB96-mediated ABA signals are integrated into an auxin signaling pathway that involves a subset of GH3 genes encoding auxin-conjugating enzymes. A MYB96-overexpressing Arabidopsis (Arabidopsis thaliana) mutant exhibited enhanced drought resistance with reduced lateral roots. In the mutant, while lateral root primordia were normally developed, meristem activation and lateral root elongation were suppressed. In contrast, a T-DNA insertional knockout mutant was more susceptible to drought. Auxin also induces MYB96 primarily in the roots, which in turn induces the GH3 genes and modulates endogenous auxin levels during lateral root development. We propose that MYB96 is a molecular link that mediates ABA-auxin cross talk in drought stress response and lateral root growth, providing an adaptive strategy under drought stress conditions.

Influence of Leaf Tolerance Mechanisms and Rain on Boron Toxicity in Barley and Wheat

Reid, R., Fitzpatrick, K. — Wed, 02 Sep 2009 10:00:33 PDT

Boron (B) toxicity is common in many areas of the world. Plant tolerance to high B varies widely and has previously been attributed to reduced uptake of B, most commonly as a result of B efflux from roots. In this study, it is shown that the expression of genes encoding B efflux transporters in leaves of wheat (Triticum aestivum) and barley (Hordeum vulgare) is associated with an ability of leaf tissues to withstand higher concentrations of B. In tolerant cultivars, necrosis in leaves occurred at B concentrations more than 2-fold higher than in sensitive cultivars. It is hypothesized that this leaf tolerance is achieved via redistribution of B by efflux transporters from sensitive symplastic compartments into the leaf apoplast. Measurements of B concentrations in leaf protoplasts, and of B released following infiltration of leaves, support this hypothesis. It was also shown that under B-toxic conditions, leaching of B from leaves by rain had a strong positive effect on growth of both roots and shoots. Measurements of rates of guttation and the concentration of B in guttation droplets indicated that the impact of guttation on the alleviation of B toxicity would be small.

Thiamin Confers Enhanced Tolerance to Oxidative Stress in Arabidopsis

Wed, 02 Sep 2009 10:00:33 PDT

Thiamin and thiamin pyrophosphate (TPP) are well known for their important roles in human nutrition and enzyme catalysis. In this work, we present new evidence for an additional role of these compounds in the protection of cells against oxidative damage. Arabidopsis (Arabidopsis thaliana) plants subjected to abiotic stress conditions, such as high light, cold, osmotic, salinity, and oxidative treatments, accumulated thiamin and TPP. Moreover, the accumulation of these compounds in plants subjected to oxidative stress was accompanied by enhanced expression of transcripts encoding thiamin biosynthetic enzymes. When supplemented with exogenous thiamin, wild-type plants displayed enhanced tolerance to oxidative stress induced by paraquat. Thiamin application was also found to protect the reactive oxygen species-sensitive ascorbate peroxidase1 mutant from oxidative stress. Thiamin-induced tolerance to oxidative stress was accompanied by decreased production of reactive oxygen species in plants, as evidenced from decreased protein carbonylation and hydrogen peroxide accumulation. Because thiamin could protect the salicylic acid induction-deficient1 mutant against oxidative stress, thiamin-induced oxidative protection is likely independent of salicylic acid signaling or accumulation. Taken together, our studies suggest that thiamin and TPP function as important stress-response molecules that alleviate oxidative stress during different abiotic stress conditions.

Characterization of New Maize Genes Putatively Involved in Cytokinin Metabolism and Their Expression during Osmotic Stress in Relation to Cytokinin Levels

Wed, 02 Sep 2009 10:00:33 PDT

Plant hormones, cytokinins (CKs), have been for a long time considered to be involved in plant responses to stress. However, their exact roles in processes linked to stress signalization and acclimatization to adverse environmental conditions are unknown. In this study, expression profiles of the entire gene families of CK biosynthetic and degradation genes in maize (Zea mays) during development and stress responses are described. Transcript abundance of particular genes is discussed in relation to the levels of different CK metabolites. Salt and osmotic stresses induce expression of some CK biosynthetic genes in seedlings of maize, leading to a moderate increase of active forms of CKs lasting several days during acclimatization to stress. A direct effect of CKs to mediate activation of stress responses does not seem to be possible due to the slow changes in metabolite levels. However, expression of genes involved in cytokinin signal transduction is uniformly down-regulated within 0.5 h of stress induction by an unknown mechanism. cis-Zeatin and its derivatives were found to be the most abundant CKs in young maize seedlings. We demonstrate that levels of this zeatin isomer are significantly enhanced during early stress response and that it originates independently from de novo biosynthesis in stressed tissues, possibly by elevated specific RNA degradation. By enhancing their CK levels, plants could perhaps undergo a reduction of growth rates maintained by abscisic acid accumulation in stressed tissues. A second role for cytokinin receptors in sensing turgor response is hypothesized besides their documented function in CK signaling.