Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant (LEA) Genes to Investigate Enhanced Stress Tolerance

In order to identify the function of late embryogenesis abundant (LEA) genes, in vitro functional analyses were perfo rmed using an Escherichia coli heterologous expression system. Three soybean late embryogenesis abundant (LEA) genes, PM11 (GenBank accession No. AF004805; group 1), PM30 (AF1 17884; group 3), and ZLDE-2 (AY351918; group 2), were cloned and expressed in a pET-28a system.The gene products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. E. coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress. Compared with control cells, the E. coli cells expressing either PM11 or PM30 showed a shorter lag period and improved growth when transferred to LB (Luria-Bertani) liquid media containing 800 mmol/L NaCl or 700 mmol/L KCl or after 4 ℃ treatment. E. coli cells expressing ZLDE-2 did not show obvious growth improvement both in either high KCl medium or after 4 ℃ treatment. The results indicate that the E. coli expression system is a simple, useful method to identify the functions of some stress-tolerant genes from plants.     

Functional Analysis of the Group 4 Late Embryogenesis Abundant Proteins Reveals Their Relevance in the Adaptive Response during Water Deficit in Arabidopsis

Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.Water deficit is a common environmental condition that leads to various responses that help in the adaptation or adjustment of an organism to this stress, considered one of the most important environmental stresses influencing plant productivity (Bray, 1997; Morison et al., 2008). The adverse effects of this environmental stress need to be counteracted, mainly because of the increasing soil desertification in cultivated and uncultivated regions. This fact demands that plants tolerate drying periods and elevated salt concentrations in the soil, which may be accompanied by extreme temperatures. Also, an interest in understanding the mechanisms by which plants sense and respond to these environmental cues accounts for the most important reasons to study in detail the responses that have been selected in plants to cope with water deficit.The acquisition of desiccation tolerance during late stages of seed development is correlated with the induction of a set of small, highly hydrophilic proteins called Late-Embryogenesis Abundant (LEA) proteins (Dure et al., 1989). These proteins are ubiquitous in plants, and although there are several classifications, we will follow that of Battaglia et al. (2008), where they are classified into seven groups on the basis of sequence similarity. Analysis of the protein sequences in these groups from different plant species defined distinctive motifs within groups (Dure, 1993; Battaglia et al., 2008). The number of members is different for each LEA protein group and varies according to the plant species. Most LEA proteins are hydrophilins, a set of proteins characterized by their biased amino acid composition, richness in Gly and other small and/or charged residues, and high hydrophilicity index (Garay-Arroyo et al., 2000). This amino acid composition promotes their flexible structure in solution, existing mainly as random coils, with the exception of the hydrophobic or atypical LEA proteins (Singh et al., 2005). Moreover, hydrophilic LEA proteins from groups 2, 3, and 4 show a prevalence of typical spectroscopic patterns of intrinsically unstructured proteins, with the occurrence of transitions from intrinsically unstructured proteins to ordered conformations in the presence of helix-promoting solvents or air drying (McCubbin et al., 1985; Russouw et al., 1995; Eom et al., 1996; Lisse et al., 1996; Ismail et al., 1999; Wolkers et al., 2001; Soulages et al., 2002, 2003; Goyal et al., 2003; Shih et al., 2004; Tolleter et al., 2007). Their high content of water-interacting residues facilitates the scavenging of water molecules, which is of special importance during developmental stages where a programmed desiccation of tissues takes place, as in the dry seed (Dure et al., 1989), or when cells experience changes in their water status (Colmenero-Flores et al., 1999). Remarkably, there is also an elevated induction in the expression of these proteins in vegetative tissues after exposure to water deficit in basically all plants that have been analyzed. In recent years, proteins with similar characteristics and expression patterns have also been detected to be induced in response to osmotic stress in bacteria and yeast (Stacy and Aalen, 1998; Garay-Arroyo et al., 2000), algae (Honjoh et al., 1995, 2000; Tanaka et al., 2004), nematodes (Solomon et al., 2000; Browne et al., 2004), rotifers (Tunnacliffe et al., 2005), and arthropods (Menze et al., 2009).One of the hypotheses regarding their function is that these proteins may act as protectors of macromolecules and/or some cellular structures during water deficit, by preferentially interacting with the available water molecules and providing a hydration shell to protect “target” integrity and function (Bray, 1997; Garay-Arroyo et al., 2000; Hoekstra et al., 2001). The use of an in vitro dehydration assay, in which the activity of malate dehydrogenase and lactate dehydrogenase was measured in the presence or absence of a hydrophilic protein, showed that plant hydrophilins (LEA proteins from groups 2, 3, and 4) and hydrophilins from Saccharomyces cerevisiae and Escherichia coli were able to protect these enzymatic activities under low water availability conditions (Reyes et al., 2005). Similarly, in vitro assays using the same or other enzymes have been used to assess the protective capacities of LEA proteins under dehydration and cold (Honjoh et al., 2000; Hara et al., 2001; Bravo et al., 2003; Goyal et al., 2005; Grelet et al., 2005; Nakayama et al., 2007; Reyes et al., 2008). In some of these assays, the ratio of LEA protein to enzyme was 1:1, suggesting that the LEA protein protective activity is not only due to the formation of a preferential hydration shell but also to an additional effect probably related to a direct interaction with their targets (Reyes et al., 2005, 2008).There are many reports of LEA proteins expressed in transgenic plants under the control of regulated or constitutive promoters, showing tolerant phenotypes under drought, high salinity, or freezing stress (Xu et al., 1996; Sivamani et al., 2000; NDong et al., 2002; Chandra Babu et al., 2004; Puhakainen et al., 2004; Fu et al., 2007; Lal et al., 2007; Xiao et al., 2007; Dalal et al., 2009). Also, the heterologous expression in bacteria and yeast of some LEA proteins confers salt and freezing tolerance (Imai et al., 1996; Zhang et al., 2000; Liu and Zheng, 2005). However, this “gain-of-function” approach does not necessarily reflect their direct participation in the plant adjustment or adaptation to these stress conditions but rather their potential to confer tolerance when ectopically expressed. In contrast, the results of a “loss-of-function” approach will lead to a direct indication of the participation of a particular gene within this process. Even though there is a large extent of information regarding the different properties of LEA proteins, our knowledge concerning their role in plant adaptation to water-limiting conditions is insufficient.In this work, we focus on the study of the group 4 LEA proteins of Arabidopsis (Arabidopsis thaliana). With only three genes in the genome (AtLEA4-1, AtLEA4-2, and AtLEA4-5), the AtLEA4 group is one of the smallest groups in Arabidopsis (Battaglia et al., 2008; Hundertmark and Hincha, 2008), which makes it accessible for a loss-of-function analysis. The LEA4 proteins are characterized by a high content of A, T, and G amino acid residues, the latter highly represented in unstructured proteins. They have a conserved N-terminal domain of 70 to 80 residues, predicted to form amphipathic α-helices, and a less conserved C-terminal region with variable size and random coil structure (Dure, 1993). Like other LEA proteins, the LEA4 group is highly accumulated in all embryo tissues of dry seeds (Roberts et al., 1993). Recently, Wise (2002) performed a bioinformatics analysis and questioned the existence of a group 4 of LEA proteins as a distinct group of LEA proteins from group 3. The algorithm used the overrepresentation/underrepresentation of particular amino acids within small motifs in the protein, giving rise to a different classification for these proteins (Wise, 2003). In support of the original classification proposed by Dure et al. (1989) and because of the high sequence conservation within this group in plants, in this work, we present genetic and functional evidence that group 4 of LEA proteins is indeed a distinct group conserved in the plant kingdom. The results reported here show that overexpression of one of the AtLEA4 proteins in Arabidopsis leads to a tolerant phenotype compared with their wild-type counterparts in their capability to endure severe water deficit and that the reduction in the accumulation levels of these proteins leads to plants more sensitive to water-limiting conditions than their wild-type genotypes. Altogether, these data constitute, to our knowledge, the first direct evidence indicating that LEA4 proteins are involved in the adaptive response of vascular plants to withstand water deficit.     

Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant （LEA） Genes to Investigate Enhanced Stress Tolerance

In order to identify the function of late embryogenesis abundant (LEA) genes, in vitro functional analyses were performed using an Escherichia coli heterologous expression system. Three soybean late embryogenesis abundant (LEA) genes, PMll (GenBank accession No. AF004805; group 1), PM30(AF117884; group 3), and ZLDE-2 (AY351918; group 2), were cloned and expressed in a pET-28a system.The gene products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. E. coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress. Compared with control cells, the E. coli cells expressing either PMll or PM30 showed a shorter lag period and improved growth when transferred to LB (Luria-Bertani) liquid media containing 800 mmol/L NaC1 or 700 mmol/L KC1 or after 4℃ treatment. E. coli cells expressing ZLDE-2 did not show obvious growth improvement both in either high KC1 medium or after 4℃ treatment. The results indicate that the E. coli expression system is a simple, useful method to identify the functions of some stress-tolerant genes from plants.     

Heterologous Expression of the Wheat Aquaporin Gene TaTIP2;2 Compromises the Abiotic Stress Tolerance of Arabidopsis thaliana

Aquaporins are channel proteins which transport water across cell membranes. We show that the bread wheat aquaporin gene TaTIP2;2 maps to the long arm of chromosome 7b and that its product localizes to the endomembrane system. The gene is expressed constitutively in both the root and the leaf, and is down-regulated by salinity and drought stress. Salinity stress induced an increased level of C-methylation within the CNG trinucleotides in the TaTIP2;2 promoter region. The heterologous expression of TaTIP2;2 in Arabidopsis thaliana compromised its drought and salinity tolerance, suggesting that TaTIP2;2 may be a negative regulator of abiotic stress. The proline content of transgenic A. thaliana plants fell, consistent with the down-regulation of P5CS1, while the expression of SOS1, SOS2, SOS3, CBF3 and DREB2A, which are all stress tolerance-related genes acting in an ABA-independent fashion, was also down-regulated. The supply of exogenous ABA had little effect either on TaTIP2;2 expression in wheat or on the phenotype of transgenic A. thaliana. The expression level of the ABA signalling genes ABI1, ABI2 and ABF3 remained unaltered in the transgenic A. thaliana plants. Thus TaTIP2;2 probably regulates the response to stress via an ABA-independent pathway(s).     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

Metabolite Profiling Reveals Abiotic Stress Tolerance in Tn5 Mutant of Pseudomonas putida

Pseudomonas is an efficient plant growth–promoting rhizobacteria (PGPR); however, intolerance to drought and high temperature limit its application in agriculture as a bioinoculant. Transposon 5 (Tn5) mutagenesis was used to generate a stress tolerant mutant from a PGPR Pseudomonas putida NBRI1108 isolated from chickpea rhizosphere. A mutant NBRI1108T, selected after screening of nearly 10,000 transconjugants, exhibited significant tolerance towards high temperature and drought. Southern hybridization analysis of EcoRI and XhoI restricted genomic DNA of NBRI1108T confirmed that it had a single Tn5 insertion. The metabolic changes in the polar and non-polar extracts of NBRI1108 and NBRI1108T were examined using ¹H, ³¹P nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Thirty six chemically diverse metabolites consisting of amino acids, fatty acids and phospholipids were identified and quantified. Insertion of Tn5 influenced amino acid and phospholipid metabolism and resulted in significantly higher concentration of aspartic acid, glutamic acid, glycinebetaine, glycerophosphatidylcholine (GPC) and putrescine in NBRI1108T as compared to that in NBRI1108. The concentration of glutamic acid, glycinebetaine and GPC increased by 34%, 95% and 100%, respectively in the NBRI1108T as compared to that in NBRI1108. High concentration of glycerophosphatidylethanolamine (GPE) and undetected GPC in NBRI1108 indicates that biosynthesis of GPE may have taken place via the methylation pathway of phospholipid biosynthesis. However, high GPC and low GPE concentration in NBRI1108T suggest that methylation pathway and phosphatidylcholine synthase (PCS) pathway of phospholipid biosynthesis are being followed in the NBRI1108T. Application of multivariate principal component analysis (PCA) on the quantified metabolites revealed clear variations in NBRI1108 and NBRI1108T in polar and non-polar metabolites. Identification of abiotic stress tolerant metabolites from the NBRI1108T suggest that Tn5 mutagenesis enhanced tolerance towards high temperature and drought. Tolerance to drought was further confirmed in greenhouse experiments with maize as host plant, where NBRI1108T showed relatively high biomass under drought conditions.     

Distinct Redox Regulation in Sub-Cellular Compartments in Response to Various Stress Conditions in Saccharomyces cerevisiae

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E _GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E _GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H⁺/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H⁺/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.     

LEAFY COTYLEDON1 Is an Essential Regulator of Late Embryogenesis and Cotyledon Identity in Arabidopsis

LEAFY COTYLEDON1 (LEC1) is an embryo defective mutation that affects cotyledon identity in Arabidopsis. Mutant cotyledons possess trichomes that are normally a leaf trait in Arabidopsis, and the cellular organization of these organs is intermediate between that of cotyledons and leaves from wild-type plants. We present several lines of evidence that indicate that the control of late embryogenesis is compromised by the mutation. First, mutant embryos are desiccation intolerant, yet embryos can be rescued before they dry to yield homozygous recessive plants that produce defective embryos exclusively. Second, although many genes normally expressed during embryonic development are active in the mutant, at least one maturation phase-specific gene is not activated. Third, the shoot apical meristem is activated precociously in mutant embryos. Fourth, in mutant embryos, several genes characteristic of postgerminative development are expressed at levels typical of wild-type seedlings rather than embryos. We conclude that postgerminative development is initiated prematurely and that embryonic and postgerminative programs operate simultaneously in mutant embryos. The pleiotropic effects of the mutation indicate that the LEC1 gene plays a fundamental role in regulating late embryogenesis. The role of LEC1 and its relationship to other genes involved in controlling late embryonic development are discussed.     

Widespread Distribution of Cell Defense against d-Aminoacyl-tRNAs

Several l-aminoacyl-tRNA synthetases can transfer a d-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme d-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of d-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by d-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), d-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between l- and d-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant d-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3–9). Recently, using a bacterium, transfer of d-tyrosine onto tRNA^Tyr was shown to occur in vivo (10).With such misacylation reactions, the resulting d-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, d-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a d-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of d-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various d-aminoacyl moieties from the 3′-end of a tRNA (4–6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13, 14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see d-amino acids, including d-tyrosine (6). In fact, in an E. coli Δdtd strain grown in the presence of 2.4 mm d-tyrosine, as much as 40% of the cellular tRNA^Tyr pool becomes esterified with d-tyrosine (10).

TABLE 1

Distribution of DTD1 and DTD2 homologs in various phylogenetic groupsHomologs of DTD1 and DTD2 were searched for using a genomic Blast analysis against complete genomes in the NCBI Database (www.ncbi.nlm.nih.gov). Values in the table are number of species. For instance, E. coli is counted only once in γ-proteobacteria despite the fact that several E. coli strains have been sequenced.

The effect of salt and temperature on the conformational changes of P1LEA‐22, a repeat unit of plant Late Embryogenesis Abundant proteins

The effect of choline chloride on the conformational dynamics of the 11‐mer repeat unit P1LEA‐22 of group 3 Late Embryogenesis Abundant (G3LEA) proteins was studied. Circular dichroism data of aqueous solutions of P1LEA‐22 revealed that the peptide favors a polyproline II (PPII) helix structure at low temperature, with increasing temperature promoting a gain of unstructured conformations. Furthermore, increases in sample FeCl₃ or choline chloride concentrations causes a gain in PPII helical structure at low temperature. The potential role of PPII structure in intrinsically disordered and G3LEA proteins is discussed, including its ability to easily access other secondary structural conformations such as α‐helix and β‐sheet, which have been observed for dehydrated G3LEA proteins. The observed effect of FeCl₃ and choline chloride salts on P1LEA‐22 suggests favorable cation interactions with the PPII helix, supporting ion sequestration as a G3LEA protein function. As choline chloride is suggested to improve salt tolerance and protect cell membrane in plants at low temperature, our results support adoption of the PPII structure as a possible damage‐preventing measure of Late Embryogenesis Abundant proteins.     

The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in Arabidopsis. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. AtRDUF1 expression is upregulated by salt treatment. Overexpression of AtRDUF1 in Arabidopsis results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of AtRDUF1 and its close homolog AtRDUF2 (atrduf1atrduf2) was hypersensitive to salt treatment. The expression levels of the stress-response genes RD29B, RD22, and KIN1 are more sensitive to salt treatment in AtRDUF1 overexpression plants. In summary, our data show that AtRDUF1 positively regulates responses to salt stress in Arabidopsis.     

Genomic Analysis of Stress Response against Arsenic in Caenorhabditis elegans

Arsenic, a known human carcinogen, is widely distributed around the world and found in particularly high concentrations in certain regions including Southwestern US, Eastern Europe, India, China, Taiwan and Mexico. Chronic arsenic poisoning affects millions of people worldwide and is associated with increased risk of many diseases including arthrosclerosis, diabetes and cancer. In this study, we explored genome level global responses to high and low levels of arsenic exposure in Caenorhabditis elegans using Affymetrix expression microarrays. This experimental design allows us to do microarray analysis of dose-response relationships of global gene expression patterns. High dose (0.03%) exposure caused stronger global gene expression changes in comparison with low dose (0.003%) exposure, suggesting a positive dose-response correlation. Biological processes such as oxidative stress, and iron metabolism, which were previously reported to be involved in arsenic toxicity studies using cultured cells, experimental animals, and humans, were found to be affected in C. elegans. We performed genome-wide gene expression comparisons between our microarray data and publicly available C. elegans microarray datasets of cadmium, and sediment exposure samples of German rivers Rhine and Elbe. Bioinformatics analysis of arsenic-responsive regulatory networks were done using FastMEDUSA program. FastMEDUSA analysis identified cancer-related genes, particularly genes associated with leukemia, such as dnj-11, which encodes a protein orthologous to the mammalian ZRF1/MIDA1/MPP11/DNAJC2 family of ribosome-associated molecular chaperones. We analyzed the protective functions of several of the identified genes using RNAi. Our study indicates that C. elegans could be a substitute model to study the mechanism of metal toxicity using high-throughput expression data and bioinformatics tools such as FastMEDUSA.     

The Distribution and Cooperation of Antioxidant (Iso)enzymes and Antioxidants in Different Subcellular Compartments in Maize Leaves during Water Stress

The effects of mild water stress induced by polyethylene glycol (PEG) on the activities of antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR)] and their isoenzymes and the antioxidant content [ascorbate (ASC) and glutathione (GSH)] of different subcellular compartments were investigated in maize. For each subcellular compartment, the activities of almost all isoenzymes resolved on native PAGE increased after 4–12 h of exposure to water stress and declined after that, showing concomitant changes with the activities of their respective total enzymes and the antioxidant content. For each subcellular compartment, at least one isoform for the detected antioxidant enzymes was resolved, but different kinds of antioxidant isoenzymes in different subcellular compartments had different responses to water stress. The relative contribution of Fe–SOD in chloroplasts and Mn–SOD in mitochondria was higher than that in other subcellular compartments. However, in apoplasts the activities of Mn–SOD and Fe–SOD declined during the process of water stress, in contrast to those located in other subcellular compartments. The results from the activities of antioxidant (iso)enzymes demonstrated that all antioxidant enzymes in all subcellular compartments were mobilized in cooperation and responded synchronously under mild water stress, with the same trend of changes in their activity. This indicated their orchestrated effects in scavenging reactive oxygen species (ROS) in situ. Additionally, the results suggested that mitochondria and apoplasts, responding most actively, might be targets for improving plant performance under mild water stress.     

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in Arabidopsis thaliana

During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of Arabidopsis thaliana. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.     

Patterns of Cell Division,Cell Differentiation and Cell Elongation in Epidermis and Cortex of Arabidopsis pedicels in the Wild Type and in erecta

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.     

Effect of Water Stress on Cell Division and Cdc2-Like Cell Cycle Kinase Activity in Wheat Leaves

In wheat (Triticum aestivum) seedlings subjected to a mild water stress (water potential of −0.3 MPa), the leaf-elongation rate was reduced by one-half and the mitotic activity of mesophyll cells was reduced to 42% of well-watered controls within 1 d. There was also a reduction in the length of the zone of mesophyll cell division to within 4 mm from the base compared with 8 mm in control leaves. However, the period of division continued longer in the stressed than in the control leaves, and the final cell number in the stressed leaves reached 85% of controls. Cyclin-dependent protein kinase enzymes that are required in vivo for DNA replication and mitosis were recovered from the meristematic zone of leaves by affinity for p13^suc1. Water stress caused a reduction in H1 histone kinase activity to one-half of the control level, although amounts of the enzyme were unaffected. Reduced activity was correlated with an increased proportion of the 34-kD Cdc2-like kinase (an enzyme sharing with the Cdc2 protein of other eukaryotes the same size, antigenic sites, affinity for p13^suc1, and H1 histone kinase catalytic activity) deactivated by tyrosine phosphorylation. Deactivation to 50% occurred within 3 h of stress imposition in cells at the base of the meristematic zone and was therefore too fast to be explained by a reduction in the rate at which cells reached mitosis because of slowing of growth; rather, stress must have acted more immediately on the enzyme. The operation of controls slowing the exit from the G1 and G2 phases is discussed. We suggest that a water-stress signal acts on Cdc2 kinase by increasing phosphorylation of tyrosine, causing a shift to the inhibited form and slowing cell production.