PLANT PHYSIOLOGY BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Quantitative 1H Nuclear Magnetic Resonance Metabolite Profiling as a Functional Genomics Platform to Investigate Alkaloid Biosynthesis in Opium Poppy

Hagel, J. M., Weljie, A. M., Vogel, H. J., Facchini, P. J. — 2008-08-04

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we used ¹H nuclear magnetic resonance (NMR) metabolite profiling for the analysis of primary and secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the ¹H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loading plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex metabolites, performed using Chenomx NMR Suite version 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other metabolites, indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine and reduced levels of sucrose, some amino acids, and malate. Real-time polymerase chain reaction analysis of 42 genes involved in primary and secondary metabolism showed differential gene expression mainly associated with alkaloid biosynthesis. Reduced alkaloid levels in the condiment variety were associated with the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The Role of Acetyl-Coenzyme A Synthetase in Arabidopsis

Lin, M., Oliver, D. J. — 2008-08-04

The acs1 knockout mutant that has a disruption in the plastidic acetyl-coenzyme A (CoA) synthetase (ACS; At5g36880) gene was used to explore the role of this protein and plastidic acetate metabolism in Arabidopsis (Arabidopsis thaliana). Disruption of the ACS gene decreased ACS activity by 90% and largely blocked the incorporation of exogenous ¹⁴C-acetate and ¹⁴C-ethanol into fatty acids. Whereas the disruption had no significant effect on the synthesis of bulk seed triacylglycerols, the acs1 plants were smaller and flowered later. This suggests that the pyruvate dehydrogenase bypass provided by the aerobic fermentation pathway that converts pyruvate to acetate and probably on to fatty acids is important to the plants during normal growth. The role of ACS in destroying fermentative intermediates is supported by the increased sensitivity of the acs1 mutant to exogenous acetate, ethanol, and acetaldehyde compared to wild-type plants. Whereas these observations suggest that flux through the aerobic fermentation pathway is important, the reason for this flux is unclear. Interestingly, acetate is able to support high rates of plant growth on medium and this growth is blocked in the acs1 mutant.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] A Proteomic Profiling Approach to Reveal a Novel Role of Brassica napus Drought 22 kD/Water-Soluble Chlorophyll-Binding Protein in Young Leaves during Nitrogen Remobilization Induced by Stressful Conditions

2008-08-04

Despite its water-soluble chlorophyll-binding protein (WSCP) function, the putative trypsin inhibitor (TI) activity of the Brassica napus drought 22 kD (BnD22) protein and its physiological function in young leaves during leaf nitrogen (N) remobilization promoted by stressful conditions remains an enigma. Therefore, our objectives were to determine (1) if BnD22 is related to the 19-kD TI previously detected in B. napus young leaves, and (2) if the levels of BnD22 transcripts, BnD22 protein, and TI activity in young leaves are associated with plant responses to stress conditions (N starvation and methyl jasmonate [MeJA] treatments) that are able to modulate leaf senescence. Compared to control, N starvation delayed initiation of senescence and induced 19-kD TI activity in the young leaves. After 3 d with MeJA, the 19-kD TI activity was 7-fold higher than the control. Using two-dimensional electrophoresis gel, TI activity, and electrospray ionization liquid chromatography tandem mass spectrometry analysis, it was demonstrated that two 19-kD proteins with isoelectric points 5.0 and 5.1 harboring TI activity correspond to BnD22 perfectly. BnD22 gene expression, TI activities, and BnD22 protein presented similar patterns. Using polyclonal anti-WSCP antibodies of Brassica oleracea, six polypeptides separated by two-dimensional electrophoresis were detected in young leaves treated with MeJA. Electrospray ionization liquid chromatography tandem mass spectrometry analysis of six polypeptides confirms their homologies with WSCP. Results suggest that BnD22 possesses dual functions (WSCP and TI) that lead to the protection of younger tissues from adverse conditions by maintaining metabolism (protein integrity and photosynthesis). By sustaining sink growth of stressed plants, BnD22 may contribute to a better utilization of recycling N from sources, a physiological trait that improves N-use efficiency.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Bridging the Gap between Plant and Mammalian Polyamine Catabolism: A Novel Peroxisomal Polyamine Oxidase Responsible for a Full Back-Conversion Pathway in Arabidopsis

2008-08-04

In contrast to animals, where polyamine (PA) catabolism efficiently converts spermine (Spm) to putrescine (Put), plants have been considered to possess a PA catabolic pathway producing 1,3-diaminopropane, ¹-pyrroline, the corresponding aldehyde, and hydrogen peroxide but unable to back-convert Spm to Put. Arabidopsis (Arabidopsis thaliana) genome contains at least five putative PA oxidase (PAO) members with yet-unknown localization and physiological role(s). AtPAO1 was recently identified as an enzyme similar to the mammalian Spm oxidase, which converts Spm to spermidine (Spd). In this work, we have performed in silico analysis of the five Arabidopsis genes and have identified PAO3 (AtPAO3) as a nontypical PAO, in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N¹-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (agmatine, cadaverine, and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C terminus, and it targets green fluorescence protein to peroxisomes when fused at the N terminus but not at the C terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis

2008-08-04

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The Transport of Sugars to Developing Embryos Is Not via the Bulk Endosperm in Oilseed Rape Seeds

2008-08-04

The fate of sucrose (Suc) supplied via the phloem to developing oilseed rape (Brassica napus) seeds has been investigated by supplying [¹⁴C]Suc to pedicels of detached, developing siliques. The method gives high, sustained rates of lipid synthesis in developing embryos within the silique comparable with those on the intact plant. At very early developmental stages (3 d after anthesis), the liquid fraction that occupies most of the interior of the seed has a very high hexose-to-Suc ratio and [¹⁴C]Suc entering the seeds is rapidly converted to hexoses. Between 3 and 12 d after anthesis, the hexose-to-Suc ratio of the liquid fraction of the seed remains high, but the fraction of [¹⁴C]Suc converted to hexose falls dramatically. Instead, most of the [¹⁴C]Suc entering the seed is rapidly converted to products in the growing embryo. These data, together with light and nuclear magnetic resonance microscopy, reveal complex compartmentation of sugar metabolism and transport within the seed during development. The bulk of the sugar in the liquid fraction of the seed is probably contained within the central vacuole of the endosperm. This sugar is not in contact with the embryo and is not on the path taken by carbon from the phloem to the embryo. These findings have important implications for the sugar switch model of embryo development and for understanding the relationship between the embryo and the surrounding endosperm.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Biochemical and Genomic Characterization of Terpene Synthases in Magnolia grandiflora

Lee, S., Chappell, J. — 2008-07-08

Magnolia grandiflora (Southern Magnolia) is a primitive evergreen tree that has attracted attention because of its horticultural distinctiveness, the wealth of natural products associated with it, and its evolutionary position as a basal angiosperm. Three cDNAs corresponding to terpene synthase (TPS) genes expressed in young leaves were isolated, and the corresponding enzymes were functionally characterized in vitro. Recombinant Mg25 converted farnesyl diphosphate (C₁₅) predominantly to β-cubebene, while Mg17 converted geranyl diphosphate (C₅) to -terpineol. Efforts to functionally characterize Mg11 were unsuccessful. Transcript levels for all three genes were prominent in young leaf tissue and significantly elevated for Mg25 and Mg11 messenger RNAs in stamens. A putative amino-terminal signal peptide of Mg17 targeted the reporter green fluorescent protein to both chloroplasts and mitochondria when transiently expressed in epidermal cells of Nicotiana tabacum leaves. Phylogenetic analyses indicated that Mg25 and Mg11 belonged to the angiosperm sesquiterpene synthase subclass TPS-a, while Mg17 aligned more closely to the angiosperm monoterpene synthase subclass TPS-b. Unexpectedly, the intron-exon organizations for the three Magnolia TPS genes were different from one another and from other well-characterized TPS gene sets. The Mg17 gene consists of six introns arranged in a manner similar to many other angiosperm sesquiterpene synthases, but Mg11 contains only four introns, and Mg25 has only a single intron located near the 5' terminus of the gene. Our results suggest that the structural diversity observed in the Magnolia TPS genes could have occurred either by a rapid loss of introns from a common ancestor TPS gene or by a gain of introns into an intron-deficient progenote TPS gene.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Inactive Methyl Indole-3-Acetic Acid Ester Can Be Hydrolyzed and Activated by Several Esterases Belonging to the AtMES Esterase Family of Arabidopsis

Yang, Y., Xu, R., Ma, C.-j., Vlot, A. C., Klessig, D. F., Pichersky, E. — 2008-07-08

The plant hormone auxin (indole-3-acetic acid [IAA]) is found both free and conjugated to a variety of carbohydrates, amino acids, and peptides. We have recently shown that IAA could be converted to its methyl ester (MeIAA) by the Arabidopsis (Arabidopsis thaliana) enzyme IAA carboxyl methyltransferase 1. However, the presence and function of MeIAA in vivo remains unclear. Recently, it has been shown that the tobacco (Nicotiana tabacum) protein SABP2 (salicylic acid binding protein 2) hydrolyzes methyl salicylate to salicylic acid. There are 20 homologs of SABP2 in the genome of Arabidopsis, which we have named AtMES (for methyl esterases). We tested 15 of the proteins encoded by these genes in biochemical assays with various substrates and identified several candidate MeIAA esterases that could hydrolyze MeIAA. MeIAA, like IAA, exerts inhibitory activity on the growth of wild-type roots when applied exogenously. However, the roots of Arabidopsis plants carrying T-DNA insertions in the putative MeIAA esterase gene AtMES17 (At3g10870) displayed significantly decreased sensitivity to MeIAA compared with wild-type roots while remaining as sensitive to free IAA as wild-type roots. Incubating seedlings in the presence of [¹⁴C]MeIAA for 30 min revealed that mes17 mutants hydrolyzed only 40% of the [¹⁴C]MeIAA taken up by plants, whereas wild-type plants hydrolyzed 100% of absorbed [¹⁴C]MeIAA. Roots of Arabidopsis plants overexpressing AtMES17 showed increased sensitivity to MeIAA but not to IAA. Additionally, mes17 plants have longer hypocotyls and display increased expression of the auxin-responsive DR5:β-glucuronidase reporter gene, suggesting a perturbation in IAA homeostasis and/or transport. mes17-1/axr1-3 double mutant plants have the same phenotype as axr1-3, suggesting MES17 acts upstream of AXR1. The protein encoded by AtMES17 had a K_m value of 13 µm and a K_cat value of 0.18 s^–1 for MeIAA. AtMES17 was expressed at the highest levels in shoot apex, stem, and root of Arabidopsis. Our results demonstrate that MeIAA is an inactive form of IAA, and the manifestations of MeIAA in vivo activity are due to the action of free IAA that is generated from MeIAA upon hydrolysis by one or more plant esterases.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Functional Analysis of a Predicted Flavonol Synthase Gene Family in Arabidopsis

2008-07-08

The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these "pseudogenes" have alternative, noncatalytic functions that have not yet been uncovered.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] A Pathogenic Fungi Diphenyl Ether Phytotoxin Targets Plant Enoyl (Acyl Carrier Protein) Reductase

Dayan, F. E., Ferreira, D., Wang, Y.-H., Khan, I. A., McInroy, J. A., Pan, Z. — 2008-07-08

Cyperin is a natural diphenyl ether phytotoxin produced by several fungal plant pathogens. At high concentrations, this metabolite inhibits protoporphyrinogen oxidase, a key enzyme in porphyrin synthesis. However, unlike its herbicide structural analogs, the mode of action of cyperin is not light dependent, causing loss of membrane integrity in the dark. We report that this natural diphenyl ether inhibits Arabidopsis (Arabidopsis thaliana) enoyl (acyl carrier protein) reductase (ENR). This enzyme is also sensitive to triclosan, a synthetic antimicrobial diphenyl ether. Whereas cyperin was much less potent than triclosan on this target site, their ability to cause light-independent disruption of membrane integrity and inhibition of ENR is similar at their respective phytotoxic concentrations. The sequence of ENR is highly conserved within higher plants and a homology model of Arabidopsis ENR was derived from the crystal structure of the protein from Brassica napus. Cyperin mimicked the binding of triclosan in the binding pocket of ENR. Both molecules were stabilized by the - stacking interaction between one of their phenyl rings and the nicotinamide ring of the NAD⁺. Furthermore, the side chain of tyrosine is involved in hydrogen bonding with a phenolic hydroxy group of cyperin. Therefore, cyperin may contribute to the virulence of the pathogens by inhibiting ENR and destabilizing the membrane integrity of the cells surrounding the point of infection.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The {beta}-Glucosidases Responsible for Bioactivation of Hydroxynitrile Glucosides in Lotus japonicus

2008-07-08

Lotus japonicus accumulates the hydroxynitrile glucosides lotaustralin, linamarin, and rhodiocyanosides A and D. Upon tissue disruption, the hydroxynitrile glucosides are bioactivated by hydrolysis by specific β-glucosidases. A mixture of two hydroxynitrile glucoside-cleaving β-glucosidases was isolated from L. japonicus leaves and identified by protein sequencing as LjBGD2 and LjBGD4. The isolated hydroxynitrile glucoside-cleaving β-glucosidases preferentially hydrolyzed rhodiocyanoside A and lotaustralin, whereas linamarin was only slowly hydrolyzed, in agreement with measurements of their rate of degradation upon tissue disruption in L. japonicus leaves. Comparative homology modeling predicted that LjBGD2 and LjBGD4 had nearly identical overall topologies and substrate-binding pockets. Heterologous expression of LjBGD2 and LjBGD4 in Arabidopsis (Arabidopsis thaliana) enabled analysis of their individual substrate specificity profiles and confirmed that both LjBGD2 and LjBGD4 preferentially hydrolyze the hydroxynitrile glucosides present in L. japonicus. Phylogenetic analyses revealed a third L. japonicus putative hydroxynitrile glucoside-cleaving β-glucosidase, LjBGD7. Reverse transcription-polymerase chain reaction analysis showed that LjBGD2 and LjBGD4 are expressed in aerial parts of young L. japonicus plants, while LjBGD7 is expressed exclusively in roots. The differential expression pattern of LjBGD2, LjBGD4, and LjBGD7 corresponds to the previously observed expression profile for CYP79D3 and CYP79D4, encoding the two cytochromes P450 that catalyze the first committed step in the biosyntheis of hydroxynitrile glucosides in L. japonicus, with CYP79D3 expression in aerial tissues and CYP79D4 expression in roots.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The Potato-Specific Apyrase Is Apoplastically Localized and Has Influence on Gene Expression, Growth, and Development

Riewe, D., Grosman, L., Fernie, A. R., Wucke, C., Geigenberger, P. — 2008-07-08

Apyrases hydrolyze nucleoside triphosphates and diphosphates and are found in all eukaryotes and a few prokaryotes. Although their enzymatic properties have been well characterized, relatively little is known regarding their subcellular localization and physiological function in plants. In this study, we used reverse genetic and biochemical approaches to investigate the role of potato (Solanum tuberosum)-specific apyrase. Silencing of the apyrase gene family with RNA interference constructs under the control of the constitutive 35S promoter led to a strong decrease in apyrase activity to below 10% of the wild-type level. This decreased activity led to phenotypic changes in the transgenic lines, including a general retardation in growth, an increase in tuber number per plant, and differences in tuber morphology. Silencing of apyrase under the control of a tuber-specific promoter led to similar changes in tuber morphology; however, there were no direct effects of apyrase inhibition on tuber metabolism. DNA microarrays revealed that decreased expression of apyrase leads to increased levels of transcripts coding for cell wall proteins involved in growth and genes involved in energy transfer and starch synthesis. To place these results in context, we determined the subcellular localization of the potato-specific apyrase. Using a combination of approaches, we were able to demonstrate that this enzyme is localized to the apoplast. We describe the evidence that underlies both this fact and that potato-specific apyrase has a crucial role in regulating growth and development.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The "Old" Euonymus europaeus Agglutinin Represents a Novel Family of Ubiquitous Plant Proteins

2008-07-08

Molecular cloning of the "old" but still unclassified Euonymus europaeus agglutinin (EEA) demonstrated that the lectin is a homodimeric protein composed of 152 residue subunits. Analysis of the deduced sequence indicated that EEA is synthesized without a signal peptide and undergoes no posttranslational processing apart from the removal of a six-residue N-terminal peptide. Glycan array screening confirmed the previously reported high reactivity of EEA toward blood group B oligosaccharides but also revealed binding to high mannose N-glycans, providing firm evidence for the occurrence of a plant carbohydrate-binding domain that can interact with structurally different glycans. Basic Local Alignment Search Tool searches indicated that EEA shares no detectable sequence similarity with any other lectin but is closely related evolutionarily to a domain that was first identified in some abscisic acid- and salt stress-responsive rice (Oryza sativa) proteins, and, according to the available sequence data, might be ubiquitous in Spermatophyta. Hence, EEA can be considered the prototype of a novel family of presumably cytoplasmic/nuclear proteins that are apparently ubiquitous in plants. Taking into account that some of these proteins are definitely stress related, the present identification of the EEA lectin domain might be a first step in the recognition of the involvement and importance of protein-glycoconjugate interactions in some essential cellular processes in Embryophyta.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] An Oleate Hydroxylase from the Fungus Claviceps purpurea: Cloning, Functional Analysis, and Expression in Arabidopsis

Meesapyodsuk, D., Qiu, X. — 2008-07-08

Claviceps purpurea, a fungal pathogen responsible for ergot diseases in many agriculturally important cereal crops, produces high levels of ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. It has been believed for many years that the biosynthesis of this fatty acid in C. purpurea involves a hydration process with linoleic acid as the substrate. Using degenerate polymerase chain reaction, we cloned a gene from the sclerotia encoding an enzyme (CpFAH) that has high sequence similarity to the C. purpurea oleate desaturase, but only low similarity to plant oleate hydroxylases. Functional analysis of CpFAH in yeast (Saccharomyces cerevisiae) indicated it acted predominantly as a hydroxylase, introducing hydroxyl groups at the 12-position of oleic acid and palmitoleic acid. As well, it showed ¹² desaturase activities on 16C and 18C monounsaturated fatty acids and, to a much lesser extent, ³ desaturase activities on ricinoleic acid. Heterologous expression of CpFAH under the guidance of a seed-specific promoter in Arabidopsis (Arabidopsis thaliana) wild-type and mutant (fad2/fae1) plants resulted in the accumulation of relatively higher levels of hydroxyl fatty acids in seeds. These data indicate that the biosynthesis of ricinoleic acid in C. purpurea is catalyzed by the fungal desaturase-like hydroxylase, and CpFAH, the first ¹² oleate hydroxylase of nonplant origin, is a good candidate for the transgenic production of hydroxyl fatty acids in oilseed crops.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

Li, F., Vallabhaneni, R., Yu, J., Rocheford, T., Wurtzel, E. T. — 2008-07-08

Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Overexpression of Poplar Cellulase Accelerates Growth and Disturbs the Closing Movements of Leaves in Sengon

2008-06-04

In this study, poplar (Populus alba) cellulase (PaPopCel1) was overexpressed in a tropical Leguminosae tree, sengon (Paraserianthes falcataria), by the Agrobacterium tumefaciens method. PaPopCel1 overexpression increased the length and width of stems with larger leaves, which showed a moderately higher density of green color than leaves of the wild type. The pairs of leaves on the transgenic plants closed more slowly during sunset than those on the wild-type plants. When main veins from each genotype were excised and placed on a paper towel, however, the leaves of the transgenic plants closed more rapidly than those of the wild-type plant. Based on carbohydrate analyses of cell walls, the leaves of the transgenic plants contained less wall-bound xyloglucan than those of the wild-type plants. In situ xyloglucan endotransglucosylase activity showed that the incorporation of whole xyloglucan, potentially for wall tightening, occurred in the parenchyma cells (motor cells) of the petiolule pulvinus attached to the main vein, although the transgenic plant incorporated less whole xyloglucan than the wild-type plant. These observations support the hypothesis that the paracrystalline sites of cellulose microfibrils are attacked by poplar cellulase, which loosens xyloglucan intercalation, resulting in an irreversible wall modification. This process could be the reason why the overexpression of poplar cellulase both promotes plant growth and disturbs the biological clock of the plant by altering the closing movements of the leaves of the plant.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Knocking Out Cytosolic Cysteine Synthesis Compromises the Antioxidant Capacity of the Cytosol to Maintain Discrete Concentrations of Hydrogen Peroxide in Arabidopsis

Lopez-Martin, M. C., Becana, M., Romero, L. C., Gotor, C. — 2008-06-04

Plant cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes involved in cysteine (Cys) biosynthesis and located in different subcellular compartments. These enzymes are made up of a complex variety of isoforms resulting in different subcellular Cys pools. To unravel the contribution of cytosolic Cys to plant metabolism, we characterized the knockout oas-a1.1 and osa-a1.2 mutants, deficient in the most abundant cytosolic OASTL isoform in Arabidopsis (Arabidopsis thaliana). Total intracellular Cys and glutathione concentrations were reduced, and the glutathione redox state was shifted in favor of its oxidized form. Interestingly, the capability of the mutants to chelate heavy metals did not differ from that of the wild type, but the mutants have an enhanced sensitivity to cadmium. With the aim of establishing the metabolic network most influenced by the cytosolic Cys pool, we used the ATH1 GeneChip for evaluation of differentially expressed genes in the oas-a1.1 mutant grown under nonstress conditions. The transcriptomic footprints of mutant plants had predicted functions associated with various physiological responses that are dependent on reactive oxygen species and suggested that the mutant was oxidatively stressed. Evidences that the mutation caused a perturbation in H₂O₂ homeostasis are that, in the knockout, H₂O₂ production was localized in shoots and roots; spontaneous cell death lesions occurred in the leaves; and lignification and guaiacol peroxidase activity were significantly increased. All these findings indicate that a deficiency of OAS-A1 in the cytosol promotes a perturbation in H₂O₂ homeostasis and that Cys is an important determinant of the antioxidative capacity of the cytosol in Arabidopsis.

[BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES] Naphthoquinone-Dependent Generation of Superoxide Radicals by Quinone Reductase Isolated from the Plasma Membrane of Soybean

Schopfer, P., Heyno, E., Drepper, F., Krieger-Liszkay, A. — 2008-06-04

Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O₂^·– by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O₂ and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH ≥ 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O₂. The possible function of this enzyme in the generation of O₂^·– and H₂O₂ at the plasma membrane of plants in vivo is discussed.