A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphataseThe riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation.
The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule bindingThe dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule-associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the domain of unknown function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain.
Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenalPhytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable.
Arabidopsis thimet oligopeptidases are redox-sensitive enzymes active in the local and systemic plant immune responseUpon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response.
Golgi-localized exo-β1,3-galactosidases involved in cell expansion and root growth in ArabidopsisPlant arabinogalactan proteins (AGPs) are a diverse group of cell surface– and wall–associated glycoproteins. Functionally important AGP glycans are synthesized in the Golgi apparatus, but the relationships among their glycosylation levels, processing, and functionalities are poorly understood. Here, we report the identification and functional characterization of two Golgi-localized exo-β-1,3-galactosidases from the glycosyl hydrolase 43 (GH43) family in Arabidopsis thaliana. GH43 loss-of-function mutants exhibited root cell expansion defects in sugar-containing growth media.
The BADC and BCCP subunits of chloroplast acetyl-CoA carboxylase sense the pH changes of the light–dark cycleAcetyl-CoA carboxylase (ACCase) catalyzes the first committed step in the de novo synthesis of fatty acids. The multisubunit ACCase in the chloroplast is activated by a shift to pH 8 upon light adaptation and is inhibited by a shift to pH 7 upon dark adaptation. Here, titrations with the purified ACCase biotin attachment domain-containing (BADC) and biotin carboxyl carrier protein (BCCP) subunits from Arabidopsis indicated that they can competently and independently bind biotin carboxylase (BC) but differ in responses to pH changes representing those in the plastid stroma during light or dark conditions.
Cucumber gibberellin 1-oxidase/desaturase initiates novel gibberellin catabolic pathwaysBioactive gibberellins (GAs) are central regulators of plant growth and development, including seed development. GA homeostasis is achieved via complex biosynthetic and catabolic pathways, whose exact activities remain to be elucidated. Here, we isolated two cDNAs from mature or imbibed cucumber seeds with high sequence similarity to known GA 3-oxidases. We found that one enzyme (designated here CsGA3ox5) has GA 3-oxidation activity. However, the second enzyme (designated CsGA1ox/ds) performed multiple reactions, including 1β-oxidation and 9,11-desaturation of GAs, but was lacking the 3-oxidation activity.
Raffinose synthase enhances drought tolerance through raffinose synthesis or galactinol hydrolysis in maize and Arabidopsis plantsRaffinose and its precursor galactinol accumulate in plant leaves during abiotic stress. RAFFINOSE SYNTHASE (RAFS) catalyzes raffinose formation by transferring a galactosyl group of galactinol to sucrose. However, whether RAFS contributes to plant drought tolerance and, if so, by what mechanism remains unclear. In this study, we report that expression of RAFS from maize (or corn, Zea mays) (ZmRAFS) is induced by drought, heat, cold, and salinity stresses. We found that zmrafs mutant maize plants completely lack raffinose and hyper-accumulate galactinol and are more sensitive to drought stress than the corresponding null-segregant (NS) plants.
The ACT domain in chloroplast precursor–phosphorylating STY kinases binds metabolites and allosterically regulates kinase activityProtein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain.
The specific molecular architecture of plant 3-hydroxy-3-methylglutaryl-CoA lyase3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life.
Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in ArabidopsisThe 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not construct distinct proteasome sub-types.
The plant cysteine oxidases from Arabidopsis thaliana are kinetically tailored to act as oxygen sensorsGroup VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors.
The chloroplast division protein ARC6 acts to inhibit disassembly of GDP-bound FtsZ2Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring.
A chemical genetic approach to engineer phototropin kinases for substrate labelingProtein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane–associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs.
Purification and functional characterization of the vacuolar malate transporter tDT from ArabidopsisThe exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes.
The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machineryThe biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo.
DENEDDYLASE1 Protein Counters Automodification of Neddylating Enzymes to Maintain NEDD8 Protein Homeostasis in ArabidopsisIn eukaryotes, the conjugation of the ubiquitin-like protein NEDD8 onto protein targets is an important post-translational modification. The best understood neddylation targets are the cullins, scaffold subunits of E3 ubiquitin ligases, where neddylation as well as deneddylation, facilitated by the protease activity of the CSN (COP9 signalosome), are required to control ubiquitin ligase assembly, function, and ultimately substrate degradation. Little is known about the role of other deneddylating enzymes besides CSN and the role of neddylation and deneddylation of their substrates.
Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPasesCopper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties.
The Structurally Plastic CH2 Domain Is Linked to Distinct Functions of Fimbrins/PlastinsFimbrins/plastins have been implicated in the generation of distinct actin structures, which are linked to different cellular processes. Historically, fimbrins/plastins were mainly considered as generating tight actin bundles. Here, we demonstrate that different members of the fimbrin/plastin family have diverged biochemically during evolution to generate either tight actin bundles or loose networks with distinct biochemical and biophysical properties. Using the phylogenetically and functionally distinct Arabidopsis fimbrins FIM4 and FIM5 we found that FIM4 generates both actin bundles and cross-linked actin filaments, whereas FIM5 only generates actin bundles.
The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the EnzymeStarch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes.
Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)Background: Arabidopsis maltodextrin disproportionating enzyme 1 (AtDPE1) plays a key role in chloroplast starch degradation.Results: Six AtDPE1 structures define the active site and reveal mechanistically relevant conformations of both the enzyme and substrate.Conclusion: Substrates are captured through loop rearrangements; the subtle deployment of active site residues controls catalysis.Significance: A molecular level understanding of the complete disproportionation cycle of AtDPE1 is presented.
A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA SequencePUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences.
Recapitulating the Structural Evolution of Redox Regulation in Adenosine 5′-Phosphosulfate Kinase from Cyanobacteria to PlantsBackground: In the plant sulfur assimilation pathway, APS kinase is a redox-regulated branch point enzyme.Results: Structural and biochemical analysis of the cyanobacterial APSK reveals an unregulated precursor of the plant enzyme.Conclusion: Protein engineering of cyanobacterial APSK recapitulates the structural development of redox control in the plant enzyme.Significance: Understanding the evolution of biochemical regulation provides insight for engineering metabolic controls.
Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine ResiduesBackground: Protein kinases that catalyze Tyr phosphorylation in plants in vivo are largely unknown.Results: CDPK/CPK-related protein kinases (CRKs) that auto/trans-phosphorylate Tyr residues and six substrates of these were identified. CRK knock-out mutants show reduced Tyr phosphorylation of β-tubulin proteins.Conclusion: CRKs can phosphorylate Tyr residues of β-tubulin and certain transcription factors.Significance: CRKs might be responsible for much of the protein Tyr phosphorylation in vivo.
Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in ChloroplastsRedox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions.