Arabidopsis thaliana expresses a second functional phytochelatin synthase.

Phytochelatins represent a major detoxifying pathway for heavy metals in plants and many other organisms. The Arabidopsis thaliana CAD1 (=AtPCS1) gene encodes a phytochelatin synthase and cad1 mutants are phytochelatin deficient and cadmium hypersensitive. The Arabidopsis genome contains a highly homologous gene, AtPCS2, of which expression and function were studied in order to understand the apparent non-redundancy of the two genes. Low constitutive AtPCS2 expression is detected in all plant organs analyzed. The AtPCS2 gene encodes a functional phytochelatin synthase as shown by expression in Saccharomyces cerevisiae and the complementation of a Schizosaccharomyces pombe phytochelatin synthase knockout strain.     

Putative N-acylphosphatidylethanolamine synthase from Arabidopsis thaliana is a lysoglycerophospholipid acyltransferase

AT1G78690, a gene found in Arabidopsis thaliana, has been reported to encode a N-acyltransferase that transfers an acyl chain from acyl-CoA to the headgroup of phosphatidylethanolamine (PE) to form N-acylphosphatidylethanolamine (N-acyl-PE). Our investigation suggests that At1g78690p is not a PE-dependent N-acyltransferase but is instead a lysoglycerophospholipid O-acyltransferase. We overexpressed AT1G78690 in Escherichia coli, extracted the cellular lipids, and identified the accumulating glycerophospholipid as acylphosphatidylglycerol (acyl-PG). Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-MS) analysis yielded [M - H](-) ions, corresponding by exact mass to acyl-PG rather than N-acyl-PE. Collision-induced dissociation mass spectrometry (MS/MS) yielded product ions consistent with acyl-PG. In addition, in vitro enzyme assays using both (32)P- and (14)C-radiolabeled substrates showed that AT1G78690 acylates 1-acyllysophosphatidylethanolamine (1-acyllyso-PE) and 1-acyllysophosphatidylglycerol (1-acyllyso-PG), but not PE or phosphatidylglycerol (PG), to form a diacylated product that co-migrates with PE and PG, respectively. We analyzed the diacylated product formed by AT1G78690 using a combination of base hydrolysis, phospholipase D treatment, ESI-MS, and MS/MS to show that AT1G78690 acylates the sn-2-position of 1-acyllyso-PE and 1-acyllyso-PG.     

The structure of YqeH. An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition

AtNOS1/AtNOA1 was identified as a nitric oxide-generating enzyme in plants, but that function has recently been questioned. To resolve issues surrounding AtNOA1 activity, we report the biochemical properties and a 2.36 A resolution crystal structure of a bacterial AtNOA1 ortholog (YqeH). Geobacillus YqeH fused to a putative AtNOA1 leader peptide complements growth and morphological defects of Atnoa1 mutant plants. YqeH does not synthesize nitric oxide from L-arginine but rather hydrolyzes GTP. The YqeH structure reveals a circularly permuted GTPase domain and an unusual C-terminal beta-domain. A small N-terminal domain, disordered in the structure, binds zinc. Structural homology among the C-terminal domain, the RNA-binding regulator TRAP, and the hypoxia factor pVHL define a recognition module for peptides and nucleic acids. TRAP residues important for RNA binding are conserved by the YqeH C-terminal domain, whose positioning is coupled to GTP hydrolysis. YqeH and AtNOA1 probably act as G-proteins that regulate nucleic acid recognition and not as nitric-oxide synthases.     

Cardiolipin synthase of Arabidopsis thaliana

Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.     

A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

A functional OGG1 homologue from Arabidopsis thaliana

One of the major mutagenic base lesions in DNA caused by exposure to reactive oxygen species is 7,8-dihydro-8-oxoguanine (8-oxoG). Genes coding for DNA repair enzymes that recognise 8-oxoG have been reported in bacteria, yeast, mammals and plants. The prokaryotic and eukaryotic genes are functional homologues but differ in their primary sequence. We have cloned, sequenced, and expressed a new Arabidopsis thaliana cDNA that shows sequence homology to the eukaryotic genes coding for 8-oxoG DNA N-glycosylases (OGG1). The 40.3-kDa enzyme it encodes (AtOGG1) introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxoG residue. In addition, AtOGG1 can form a Schiff base with 8-oxoG in the presence of NaBH₄, suggesting that it is a bifunctional DNA N-glycosylase. Furthermore, expression of AtOGG1 in an Escherichia coli strain that is deficient in the repair of 8-oxoG in DNA suppresses its spontaneous-mutator phenotype. Thus, we have demonstrated that AtOGG1 is not only a structural but also a functional eukaryotic OGG1 homologue.     

Phototropin from Chlamydomonas reinhardtii is functional in Arabidopsis thaliana

Phototropin, a plant blue light photoreceptor, mediates important blue light responses such as phototropism, chloroplast positioning and stomatal opening in higher plants. In Arabidopsis thaliana, two phototoropins, phototropin 1 and 2, are known. Recently, in the unicellular green alga, Chlamydomonas reinhardtii, a phototropin homolog was identified. It exhibits photochemical properties similar to those of higher plant phototropins and is involved in multiple steps of the sexual life cycle of Chlamydomonas. Here, we expressed Chlamydomonas phototropin in Arabidopsis to examine whether it is active in a distantly related plant species. The Arabidopsis mutant deficient in both phototropin 1 and 2 was transformed with a vector containing Chlamydomonas phototropin cDNA fused to a cauliflower mosaic virus 35S promoter. The resulting lines were classified into high, medium and low expressers based on RNA gel blot and immunoblot analyses. Typical phototropin responses were restored in high expression lines. These results demonstrate that Chlamydomonas phototropin is functional in higher plants. Hence, the basic mechanism of phototropin action is highly conserved, even though its apparent physiological functions are quite diverse.     

POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis

In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane-located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.     

The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase

Apocarotenoids resulting from the oxidative cleavage of carotenoids serve as important signaling and accessory molecules in a variety of biological processes. The enzymes catalyzing these reactions are referred to as carotenases or carotenoid oxygenases. Whether they act according to a monooxygenase mechanism, requiring two oxygens from different sources, or a dioxygenase mechanism is still a topic of controversy. In this study, we utilized the readily available beta-apo-8'-carotenal as a substrate for the heterologously expressed AtCCD1 protein from Arabidopsis thaliana to investigate the oxidative cleavage mechanism of the 9,10 double bond of carotenoids. Beta-ionone and a C(17)-dialdehyde were detected as products by gas and liquid chromatography-mass spectrometry as well as NMR analysis. Labeling experiments using H(2)(18)O or (18) O(2) showed that the oxygen in the keto-group of beta-ionone is derived solely from molecular dioxygen. When experiments were performed in an (18)O(2)-enriched atmosphere, a substantial fraction of the C(17)-dialdehyde contained labeled oxygen. The results unambiguously demonstrate a dioxygenase mechanism for the carotenase AtCCD1 from A. thaliana.     

An OGG1 orthologue encoding a functional 8-oxoguanine DNA glycosylase/lyase in Arabidopsis thaliana

Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsisthaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate characteristic of all bifunctional DNA glycosylases/lyases. Consistent with its in vitro activity, expression of AtOGG1 suppresses the mutator phenotype of an E. coli strain deficient in 8-oxoG repair. Our results suggest that AtOgg1 is an structural and functional homologue of Ogg1 and establish the presence of two distinct 8-oxoG repair enzymes in Arabidopsis.     

Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3

We report here the isolation of the Arabidopsis thaliana gene AtK-1. The predicted protein sequence of AtK-1 show 70% identity to the Arabidopsis ASK and alfalfa MsK kinases that are homologs of the Drosophila shaggy and rat GSK-3 serine/threonine protein kinases playing an important role in signal transduction processes in animals. Northern analysis of different organs revealed exclusive expression in inflorescences suggesting an involvement of the AtK-1 kinase in reproduction-specific processes.     

Arabidopsis thaliana GLX2-1 contains a dinuclear metal binding site, but is not a glyoxalase 2

In an effort to probe the structure and function of a predicted mitochondrial glyoxalase 2, GLX2-1, from Arabidopsis thaliana, GLX2-1 was cloned, overexpressed, purified and characterized using metal analyses, kinetics, and UV-visible, EPR, and (1)H-NMR spectroscopies. The purified enzyme was purple and contained substoichiometric amounts of iron and zinc; however, metal-binding studies reveal that GLX2-1 can bind nearly two equivalents of either iron or zinc and that the most stable analogue of GLX2-1 is the iron-containing form. UV-visible spectra of the purified enzyme suggest the presence of Fe(II) in the protein, but the Fe(II) can be oxidized over time or by the addition of metal ions to the protein. EPR spectra revealed the presence of an anti-ferromagnetically-coupled Fe(III)Fe(II) centre and the presence of a protein-bound high-spin Fe(III) centre, perhaps as part of a FeZn centre. No paramagnetically shifted peaks were observed in (1)H-NMR spectra of the GLX2-1 analogues, suggesting low amounts of the paramagnetic, anti-ferromagnetically coupled centre. Steady-state kinetic studies with several thiolester substrates indicate that GLX2-1 is not a GLX2. In contrast with all of the other GLX2 proteins characterized, GLX2-1 contains an arginine in place of one of the metal-binding histidine residues at position 246. In order to evaluate further whether Arg(246) binds metal, the R246L mutant was prepared. The metal binding results are very similar to those of native GLX2-1, suggesting that a different amino acid is recruited as a metal-binding ligand. These results demonstrate that Arabidopsis GLX2-1 is a novel member of the metallo-beta-lactamase superfamily.     

Serpin1 of Arabidopsis thaliana is a suicide inhibitor for metacaspase 9

Metacaspases are distant relatives of animal caspases found in plants, fungi and protozoa. We demonstrated previously that two type II metacaspases of Arabidopsis thaliana, AtMC4 and AtMC9 are Arg/Lys-specific cysteine-dependent proteases. We screened a combinatorial tetrapeptide library of 130,321 substrates with AtMC9. Here, we show that AtMC9 is a strict Arg/Lys-specific protease. Based on the position-specific scoring matrix derived from the substrate library results, the tetrapeptide Val-Arg-Pro-Arg was identified as an optimized substrate. AtMC9 had a kcat/KM of 4.6x10(5) M-1 s-1 for Ac-Val-Arg-Pro-Arg-amido-4-methyl-coumarin, representing a more than 10-fold improvement over existing fluorogenic substrates. A yeast two-hybrid screen with catalytically inactive AtMC9 as bait identified a serine protease inhibitor, designated AtSerpin1, which was found to be a potent inhibitor of AtMC9 activity in vitro through cleavage of its reactive center loop and covalent binding to AtMC9. On the basis of the substrate profiling of AtMC9 and confirmation through site-directed mutagenesis, the inhibitory P4-P1 cleavage site of AtSerpin1 was determined to be Ile-Lys-Leu-Arg351. Further mutagenesis of the AtSerpin1 inhibitory cleavage site modulated AtMC9 inhibition positively or negatively. Both AtMC9 and AtSerpin1 were localized in the extracellular space, suggesting an in vivo interaction as well. To our knowledge, this is the first report of plant protease inhibition by a plant serpin.     

Arabidopsis thaliana as a model for functional nectary analysis

Nectaries and nectar have received much research attention for well over 200 years due to their central roles in plant–pollinator interactions. Despite this, only a few genes have demonstrated impacts on nectary development, and none have been reported to mediate de novo nectar production. This scarcity of information is largely due to the lack of a model that combines sizeable nectaries, and high levels of nectar production, along with suitable genomics resources. For example, even though Arabidopsis thaliana has been useful for developmental studies, it has been largely overlooked as a model for studying nectary function due to the small size of its flowers. However, Arabidopsis nectaries, along with those of related species, are quite operational and can be used to discern molecular mechanisms of nectary form and function. A current understanding of the machinery underlying nectary function in plants is briefly presented, with emphasis placed on the prospects of using Arabidopsis as a model for studying these processes.