Heterologous Expression and Characterization of Plant Wax Ester Producing Enzymes.

Wax esters are widely distributed among microbes, plants, and mammals, and they serve protective and energy storage functions. Three classes of enzymes catalyze the reaction between a fatty acyl alcohol and a fatty acyl-CoA, generating wax esters. Multiple isozymes of two of these enzyme classes, the membrane-bound O-acyltransferase class of wax synthase (WS) and the bifunctional wax synthase/diacylglycerol acyl transferase (WSD), co-exist in plants. Although WSD enzymes are known to produce the wax esters of the plant cuticle, the functionality of plant WS enzymes is less well characterized. In this study, we investigated the phylogenetic relationships among the 12 WS and 11 WSD isozymes that occur in Arabidopsis, and established two in vivo heterologous expression systems, in the yeast Saccharomyces cerevisiae and in Arabidopsis seeds to investigate the catalytic abilities of the WS enzymes. These two refactored wax assembly chassis were used to demonstrate that WS isozymes show distinct differences in the types of esters that can be assembled. We also determined the cellular and subcellular localization of two Arabidopsis WS isozymes. Additionally, using publicly available Arabidopsis transcriptomics data, we identified the co-expression modules of the 12 Arabidopsis WS coding genes. Collectively, these analyses suggest that WS genes may function in cuticle assembly and in supporting novel photosynthetic function(s).

Maize Glossy2 and Glossy2-like Genes Have Overlapping and Distinct Functions in Cuticular Lipid Deposition.

Plant epidermal cells express unique molecular machinery that juxtapose the assembly of intracellular lipid components and the unique extracellular cuticular lipids that are unidirectionally secreted to plant surfaces. In maize (Zea mays), mutations at the glossy2 (gl2) locus affect the deposition of extracellular cuticular lipids. Sequence-based genome scanning identified a new Gl2 homolog in the maize genome, namely Gl2-like Both the Gl2-like and Gl2 genes are members of the BAHD superfamily of acyltransferases, with close sequence similarity to the Arabidopsis (Arabidopsis thaliana) CER2 gene. Transgenic experiments demonstrated that Gl2-like and Gl2 functionally complement the Arabidopsis cer2 mutation, with differential influences on the cuticular lipids and the lipidome of the plant, particularly affecting the longer alkyl chain acyl lipids, especially at the 32-carbon chain length. Site-directed mutagenesis of the putative BAHD catalytic HXXXDX-motif indicated that Gl2-like requires this catalytic capability to fully complement the cer2 function, but Gl2 can accomplish complementation without the need for this catalytic motif. These findings demonstrate that Gl2 and Gl2-like overlap in their cuticular lipid function, but have evolutionarily diverged to acquire nonoverlapping functions.

Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis.

The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate-dependent decarboxylating enzyme that uses branched-chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched-chain amino acids), isotope labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate-dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.

Microbial production of bi-functional molecules by diversification of the fatty acid pathway.

Fatty acids that are chemically functionalized at their ω-ends are rare in nature yet offer unique chemical and physical properties with wide ranging industrial applications as feedstocks for bio-based polymers, lubricants and surfactants. Two enzymatic determinants control this ω-group functionality, the availability of an appropriate acyl-CoA substrate for initiating fatty acid biosynthesis, and a fatty acid synthase (FAS) variant that can accommodate that substrate in the initial condensation reaction of the process. In Type II FAS, 3-ketoacyl-ACP synthase III (KASIII) catalyses this initial condensation reaction. We characterized KASIIIs from diverse bacterial sources, and identified variants with novel substrate specificities towards atypical acyl-CoA substrates, including 3-hydroxybutyryl-CoA. Using Alicyclobacillus acidocaldarius KASIII, we demonstrate the in vivo diversion of FAS to produce novel ω-1 hydroxy-branched fatty acids from glucose in two bioengineered microbial hosts. This study unveils the biocatalytic potential of KASIII for synthesizing diverse ω-functionalized fatty acids.

Zebrafish RNase T2 genes and the evolution of secretory ribonucleases in animals.

BACKGROUND: Members of the Ribonuclease (RNase) T2 family are common models for enzymological studies, and their evolution has been well characterized in plants. This family of acidic RNases is widespread, with members in almost all organisms including plants, animals, fungi, bacteria and even some viruses. While several biological functions have been proposed for these enzymes in plants, their role in animals is unknown. Interestingly, in vertebrates most of the biological roles of plant RNase T2 proteins are carried out by members of a different family, RNase A. Still, RNase T2 proteins are conserved in these animals RESULTS: As a first step to shed light on the role of animal RNase T2 enzymes, and to understand the evolution of these proteins while co-existing with the RNase A family, we characterized RNase Dre1 and RNase Dre2, the two RNase T2 genes present in the zebrafish (Danio rerio) genome. These genes are expressed in most tissues examined, including high expression in all stages of embryonic development, and their expression corresponds well with the presence of acidic RNase activities in every tissue analyzed. Embryo expression seems to be a conserved characteristic of members of this family, as other plant and animal RNase T2 genes show similar high expression during embryo development. While plant RNase T2 proteins and the vertebrate RNase A family show evidences of radiation and gene sorting, vertebrate RNase T2 proteins form a monophyletic group, but there is also another monophyletic group defining a fish-specific RNase T2 clade. CONCLUSION: Based on gene expression and phylogenetic analyses we propose that RNase T2 enzymes carry out a housekeeping function. This conserved biological role probably kept RNase T2 enzymes in animal genomes in spite of the presence of RNases A. A hypothetical role during embryo development is also discussed.

Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses.

Reactive oxygen species (ROS) play a key signaling role in plants and are controlled in cells by a complex network of ROS metabolizing enzymes found in several different cellular compartments. To study how different ROS signals, generated in different cellular compartments, are integrated in cells, we generated a double mutant lacking thylakoid ascorbate peroxidase (tylapx) and cytosolic ascorbate peroxidase1 (apx1). Our analysis suggests that two different signals are generated in plants lacking cytosolic APX1 or tylAPX. The lack of a chloroplastic hydrogen peroxide removal enzyme triggers a specific signal in cells that results in enhanced tolerance to heat stress, whereas the lack of a cytosolic hydrogen peroxide removal enzyme triggers a different signal, which results in stunted growth and enhanced sensitivity to oxidative stress. When the two signals are coactivated in cells (i.e. tylapx/apx1), a new response is detected, suggesting that the integration of the two different signals results in a new signal that manifests in late flowering, low protein oxidation during light stress, and enhanced accumulation of anthocyanins. Our results demonstrate a high degree of plasticity in ROS signaling in Arabidopsis (Arabidopsis thaliana) and suggest the existence of redundant pathways for ROS protection that compensate for the lack of classical ROS removal enzymes such as cytosolic and chloroplastic APXs. Further investigation of the enhanced heat tolerance in plants lacking tylAPX, using mutants deficient in chloroplast-to-nuclei retrograde signaling, suggests the existence of a chloroplast-generated stress signal that enhances basal thermotolerance in plants.

Enhanced tolerance to environmental stress in transgenic plants expressing the transcriptional coactivator multiprotein bridging factor 1c.

Abiotic stresses cause extensive losses to agricultural production worldwide. Acclimation of plants to abiotic conditions such as drought, salinity, or heat is mediated by a complex network of transcription factors and other regulatory genes that control multiple defense enzymes, proteins, and pathways. Associated with the activity of different transcription factors are transcriptional coactivators that enhance their binding to the basal transcription machinery. Although the importance of stress-response transcription factors was demonstrated in transgenic plants, little is known about the function of transcriptional coactivators associated with abiotic stresses. Here, we report that constitutive expression of the stress-response transcriptional coactivator multiprotein bridging factor 1c (MBF1c) in Arabidopsis (Arabidopsis thaliana) enhances the tolerance of transgenic plants to bacterial infection, heat, and osmotic stress. Moreover, the enhanced tolerance of transgenic plants to osmotic and heat stress was maintained even when these two stresses were combined. The expression of MBF1c in transgenic plants augmented the accumulation of a number of defense transcripts in response to heat stress. Transcriptome profiling and inhibitor studies suggest that MBF1c expression enhances the tolerance of transgenic plants to heat and osmotic stress by partially activating, or perturbing, the ethylene-response signal transduction pathway. Present findings suggest that MBF1 proteins could be used to enhance the tolerance of plants to different abiotic stresses.

Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis.

Reactive oxygen species (ROS), such as O2- and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2- and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1.

Measuring programmed cell death in plants.

Methods for the detection of programmed cell death (PCD) in plants are reviewed with references for different biochemical, microscopic, and molecular assays. A detailed description of three different methods for the detection of biotic or abiotic PCD in plant tissues is included. The reader is encouraged to use all three methods in parallel to obtain a reliable measure of PCD. Critical considerations are highlighted.

When defense pathways collide. The response of Arabidopsis to a combination of drought and heat stress.

Within their natural habitat, plants are subjected to a combination of abiotic conditions that include stresses such as drought and heat. Drought and heat stress have been extensively studied; however, little is known about how their combination impacts plants. The response of Arabidopsis plants to a combination of drought and heat stress was found to be distinct from that of plants subjected to drought or heat stress. Transcriptome analysis of Arabidopsis plants subjected to a combination of drought and heat stress revealed a new pattern of defense response in plants that includes a partial combination of two multigene defense pathways (i.e. drought and heat stress), as well as 454 transcripts that are specifically expressed in plants during a combination of drought and heat stress. Metabolic profiling of plants subjected to drought, heat stress, or a combination of drought and heat stress revealed that plants subject to a combination of drought and heat stress accumulated sucrose and other sugars such as maltose and glucose. In contrast, Pro that accumulated in plants subjected to drought did not accumulate in plants during a combination of drought and heat stress. Heat stress was found to ameliorate the toxicity of Pro to cells, suggesting that during a combination of drought and heat stress sucrose replaces Pro in plants as the major osmoprotectant. Our results highlight the plasticity of the plant genome and demonstrate its ability to respond to complex environmental conditions that occur in the field.

The zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis.

Cytosolic ascorbate peroxidase 1 (Apx1) is a key H(2)O(2) removal enzyme in plants. Microarray analysis of Apx1-deficient Arabidopsis plants revealed that the expression of two zinc finger proteins (Zat12 and Zat7) and a WRKY transcription factor (WRKY25) is elevated in knock-out Apx1 plants grown under controlled conditions. Because mutants lacking Apx1 accumulate H(2)O(2), we examined the correlation between H(2)O(2) and the expression of Zat12, Zat7, WRKY25, and Apx1. The expression of Zat12, Zat7, and WRKY25 was simultaneously elevated in cells in response to oxidative stress (i.e. H(2)O(2) or paraquat application), heat shock, or wounding. In contrast, light or osmotic stress did not enhance the expression of these putative transcription factors. All stresses tested enhanced the expression of Apx1. Transgenic plants expressing Zat12 or Zat7 could tolerate oxidative stress. In contrast, transgenic plants expressing WRKY25 could not. Although the expression of Zat12, Zat7, or WRKY25 in transgenic plants did not enhance the expression of Apx1 under controlled conditions, Zat12-deficient plants were unable to enhance the expression of Apx1, Zat7, or WRKY25 in response to H(2)O(2) or paraquat application. Zat12-deficient plants were also more sensitive than wild type plants to H(2)O(2) application as revealed by a higher level of H(2)O(2)-induced protein oxidation detected in these plants by protein blots. Our results suggest that Zat12 is an important component of the oxidative stress response signal transduction network of Arabidopsis required for Zat7, WRKY25, and Apx1 expression during oxidative stress.

The water-water cycle is essential for chloroplast protection in the absence of stress.

Maintaining electron flow through the photosynthetic apparatus, even in the absence of a sufficient amount of NADP+ as an electron acceptor, is essential for chloroplast protection from photooxidative stress. At least two different pathways are thought to participate in this process, i.e. cyclic electron flow and the water-water cycle. Although the function of the water-water cycle was inferred from a number of biochemical and physiological studies, genetic evidence for the function of this cycle is very limited. Here we show that knockdown Arabidopsis plants with suppressed expression of the key water-water cycle enzyme, thylakoid-attached copper/zinc superoxide dismutase (KD-SOD), are suppressed in their growth and development. Chloroplast size, chlorophyll content, and photosynthetic activity were also reduced in KD-SOD plants. Microarray analysis of KD-SOD plants, grown under controlled conditions, revealed changes in transcript expression consistent with an acclimation response to light stress. Although a number of transcripts involved in the defense of plants from oxidative stress were induced in KD-SOD plants, and seedlings of KD-SOD plants were more tolerant to oxidative stress, these mechanisms were unable to compensate for the suppression of the water-water cycle in mature leaves. Thus, the localization of copper/zinc superoxide dismutase at the vicinity of photosystem I may be essential for its function. Our studies provide genetic evidence for the importance of the water-water cycle in protecting the photosynthetic apparatus of higher plants from photooxidative damage.

Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase.

The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.

The combined effect of drought stress and heat shock on gene expression in tobacco.

In nature, plants encounter a combination of environmental conditions that may include stresses such as drought or heat shock. Although drought and heat shock have been extensively studied, little is known about how their combination affect plants. We used cDNA arrays, coupled with physiological measurements, to study the effect of drought and heat shock on tobacco (Nicotiana tabacum) plants. A combination of drought and heat shock resulted in the closure of stomata, suppression of photosynthesis, enhancement of respiration, and increased leaf temperature. Some transcripts induced during drought, e.g. those encoding dehydrin, catalase, and glycolate oxidase, and some transcripts induced during heat shock, e.g. thioredoxin peroxidase, and ascorbate peroxidase, were suppressed during a combination of drought and heat shock. In contrast, the expression of other transcripts, including alternative oxidase, glutathione peroxidase, phenylalanine ammonia lyase, pathogenesis-related proteins, a WRKY transcription factor, and an ethylene response transcriptional co-activator, was specifically induced during a combination of drought and heat shock. Photosynthetic genes were suppressed, whereas transcripts encoding some glycolysis and pentose phosphate pathway enzymes were induced, suggesting the utilization of sugars through these pathways during stress. Our results demonstrate that the response of plants to a combination of drought and heat shock, similar to the conditions in many natural environments, is different from the response of plants to each of these stresses applied individually, as typically tested in the laboratory. This response was also different from the response of plants to other stresses such as cold, salt, or pathogen attack. Therefore, improving stress tolerance of plants and crops may require a reevaluation, taking into account the effect of multiple stresses on plant metabolism and defense.