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Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. Soraphen A, a macrocyclic polyketide natural product, is a nanomolar inhibitor against the biotin carboxylase (BC) domain of human, yeast, and other eukaryotic ACCs. Here we report the crystal structures of the yeast BC domain, alone and in complex with soraphen A. Soraphen has extensive interactions with an allosteric site, about 25 A from the active site. The specificity of soraphen is explained by large structural differences between the eukaryotic and prokaryotic BC in its binding site, confirmed by our studies on the effects of single-site mutations in this binding site. Unexpectedly, our structures suggest that soraphen may bind in the BC dimer interface and inhibit the BC activity by disrupting the oligomerization of this domain. Observations from native gel electrophoresis confirm this structural insight. The structural information provides a foundation for structure-based design of new inhibitors against these enzymes.

That's all fine and good for cyanobacterial phytochrome, but the real excitement has to do with potential functional homology of plant phytochromes. Specifically, do plant phytochromes possess histidine kinase activity and what then do we make of phytochrome-associated serine kinase activity? As discussed by Quail 1997xQuail, P.H. BioEssay. 1997; 19: 571–579Crossref | PubMedSee all ReferencesQuail 1997, while there is presently no experimental evidence supporting the hypothesis that higher plant phytochromes are histidine kinases, this question must still be considered unresolved. In any case, whether or not they have histidine kinase activity, it is clear from sequence analysis and the discovery of Cph1 that plant phytochromes are evolutionarily related to histidine kinases. Given this, and assuming that phytochrome-associated serine kinase activity is intrinsic to the photoreceptor, there are two general models that seem most probable in accounting for serine kinase activity in a histidine kinase–related protein. The first model postulates that plant phytochromes, and perhaps other proteins like SpoIIAB and the mitochondrial protein kinases, represent nonorthodox members of the sensor kinase family that have altered their substrate specificity from histidines to serines (Figure 2AFigure 2A). In this case, the phosphorylated serines would be expected to be stable regulatory modifications rather than intermediates in phosphotransfer reactions due to the comparatively low energy of a phosphoester bond. As discussed previously, phytochrome autophosphorylation would presumably be a down-regulation mechanism; however, this would not preclude the existence of other substrates for phytochrome kinase activity. In this model then, phytochromes would be functionally similar to the eukaryotic protein serine/threonine/tyrosine kinase family even though they are evolutionarily related to histidine kinases.Figure 2Models of Phytochrome Kinase Activity(A) TKD1 and/or TKD2 directly catalyze the ATP-dependent phosphorylation of an N-terminal serine resulting in the down-regulation of phytochrome activity. A possible mechanism for this down-regulation would be inhibiting phytochrome kinase activity toward a subsequent signaling component X.(B) TKD1 and/or TKD2 exhibit true histidine kinase activity and catalyze the ATP-dependent phosphorylation of a nonconsensus phytochrome histidine residue. The phosphate moiety is subsequently transferred to an N-terminal serine resulting in the down-regulation of phytochrome activity. A possible mechanism for this down-regulation would be by inhibiting phosphotransfer to a response regulator X involved in the signaling pathway.View Large Image | View Hi-Res Image | Download PowerPoint SlideThe alternative model is that plant phytochromes are true histidine kinases that autophosphorylate on a nonconsensus histidine, but the phosphate moiety is then transferred to the N-terminal serine residue (Figure 2BFigure 2B). As previously noted, (Quail 1997xQuail, P.H. BioEssay. 1997; 19: 571–579Crossref | PubMedSee all ReferencesQuail 1997) there are examples of sensor kinases like CheA that autophosphorylate on nonconsensus histidines. Furthermore, there is precedence for the proposed histidine to serine phosphotransfer reaction. CheY mutants lacking the phosphate-accepting aspartate are phosphorylated by CheA on a hydroxy amino acid presumably due to proximity and the high phosphotransfer potential of a phosphohistidine (Bourret et al. 1990xBourret, R.B, Hess, F, and Simon, M.I. Proc. Natl. Acad. Sci. USA. 1990; 87: 41–45Crossref | PubMedSee all ReferencesBourret et al. 1990). One would predict that such a reaction could be favored if an internal hydroxy amino acid were properly spaced and configured relative to a phosphorylated histidine residue. In this regard, we note that the serine phosphorylated in plant phytochromes resides in a 60–100 amino acid N-terminal extension not found in Cph1 (Figure 1DFigure 1D), suggesting that this domain has a function unique to eukaryotic phytochromes. One could envision this model working either with or without a conventional response regulator. In either case, we once again postulate that phytochrome autophosphorylation would serve as a down-regulation mechanism. If response regulators were not involved in signaling, this reaction might simply be an internal feedback mechanism that may or may not have higher levels of regulation beyond phytochrome photoconversion. If response regulators were involved in phytochrome signaling, the N-terminal serine might be acting as an alternate substrate when the response regulator was not available (e.g., due to its subsequent signaling function) or when it was already phosphorylated (Figure 2BFigure 2B). Presently, the proposed histidine-to-serine phosphotransfer reaction in this model can be tested by systematically mutating all phytochrome histidine residues and assaying for an effect on N-terminal serine phosphorylation. Alternatively, the postulated histidine autophosphorylation may be more readily detected in phytochromes where the known serine phosphorylation site was mutated.In summary, we believe that the recurring connections between phytochromes and protein kinases cannot be coincidental and must be indicative of functional importance. Although it is clear that plant phytochromes arose from an ancestral prokaryotic histidine kinase, the fact that they seem to exhibit serine kinase activity indicates that they are atypical members of this superfamily. Therefore, the extent of functional homology between plant phytochromes and histidine kinases is still in question. Resolution of these ambiguities will mark an important step in our understanding of phytochrome function. This obviously is a required step if phytochrome signal transduction involves phosphorylation of subsequent pathway components.

Hepatitis C is a chronic liver disease that contributes to progressive metabolic dysfunction. Infection of hepatocytes by hepatitis C virus (HCV) results in reprogramming of hepatic and serum lipids. However, the specific contribution of these distinct pools of lipids to HCV infection remains ill defined. In this study, we investigated the role of hepatic lipogenesis in HCV infection by targeting the rate-limiting step in this pathway, which is catalyzed by the acetyl-CoA carboxylase (ACC) enzymes. Using two structurally unrelated ACC inhibitors, we determined that blockade of lipogenesis resulted in reduced viral replication, assembly, and release. Supplementing exogenous lipids to cells treated with ACC inhibitors rescued HCV assembly with no effect on viral replication and release. Intriguingly, loss of viral RNA was not recapitulated at the protein level and addition of 2-bromopalmitate, a competitive inhibitor of protein palmitoylation, mirrored the effects of ACC inhibitors on reduced viral RNA without a concurrent loss in protein expression. These correlative results suggest that newly synthesized lipids may have a role in protein palmitoylation during HCV infection.

Plant root system architecture (RSA) is plastic and dynamic, allowing plants to respond to their environment in order to optimize acquisition of important soil resources. A number of RSA traits are known to be correlated with improved crop performance. There is increasing recognition that future gains in productivity, especially under low input conditions, can be achieved through optimization of RSA. However, realization of this goal has been hampered by low resolution and low throughput approaches for characterizing RSA. To overcome these limitations, new methods are being developed to facilitate high throughput and high content RSA phenotyping. Here we summarize laboratory and field approaches for phenotyping RSA, drawing particular attention to recent advances in plant imaging and analysis. Improvements in phenotyping will facilitate the genetic analysis of RSA and aid in the identification of the genetic loci underlying useful agronomic traits.

Root system architecture (RSA) describes the dynamic spatial configuration of different types and ages of roots in a plant, which allows adaptation to different environments. Modifications in RSA enhance agronomic traits in crops and have been implicated in soil organic carbon content. Together, these fundamental properties of RSA contribute to the net carbon balance and overall sustainability of biofuels. In this article, we will review recent data supporting carbon sequestration by biofuel crops, highlight current progress in studying RSA, and discuss future opportunities for optimizing RSA for biofuel production and soil carbon sequestration.

Acetyl-CoA carboxylase (ACC) catalyses the first step in fatty-acid biosynthesis. Owing to its role in primary metabolism, ACC has been exploited as a commercial herbicide target and identified as a chemically validated fungicide target. In animals, ACC is also a key regulator of fat metabolism. This function has made ACC a prime target for the development of anti-obesity and anti-Type II diabetes therapeutics. Despite its economic importance, there is a lack of published information on recombinant expression of ACC. We report here the expression of enzymically active fungal (Ustilago maydis) ACC in Escherichia coli. The recombinant enzyme exhibited Km values of 0.14±0.013 mM and 0.19±0.041 mM for acetyl-CoA and ATP respectively, which are comparable with those reported for the endogenous enzyme. The polyketide natural product soraphen is a potent inhibitor of the BC (biotin carboxylase) domain of endogenous fungal ACC. Similarly, recombinant ACC activity was inhibited by soraphen with a Ki of 2.1±0.9 nM. A truncated BC domain that included amino acids 2–560 of the full-length protein was also expressed in E. coli. The isolated BC domain was expressed to higher levels, and was more stable than full-length ACC. Although incapable of enzymic turnover, the BC domain exhibited high-affinity soraphen binding (Kd 1.1±0.3 nM), demonstrating a native conformation. Additional BC domains from the phytopathogenic fungi Magnaporthe grisea and Phytophthora infestans were also cloned and expressed, and were shown to exhibit high-affinity soraphen binding. Together, these reagents will be useful for structural studies and assay development.

Plants constantly monitor their light environment in order to grow and develop optimally, in part through use of the phytochromes, which sense red/far-red light. A phytochrome binding protein, PKS1 (phytochrome kinase substrate 1), was identified that is a substrate for light-regulated phytochrome kinase activity in vitro. In vivo experiments suggest that PKS1 is phosphorylated in a phytochrome-dependent manner and negatively regulates phytochrome signaling. The data suggest that phytochromes signal by serine-threonine phosphorylation.

Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype.

Plant growth and development are regulated by interactions between the environment and endogenous developmental programs. Of the various environmental factors controlling plant development, light plays an especially important role, in photosynthesis, in seasonal and diurnal time sensing, and as a cue for altering developmental pattern. Recently, several laboratories have devised a variety of genetic screens using Arabidopsis thaliana to dissect the signal transduction pathways of the various photoreceptor systems. Genetic analysis demonstrates that light responses are not simply endpoints of linear signal transduction pathways but are the result of the integration of information from a variety of photoreceptors through a complex network of interacting signaling components. These signaling components include the red/far-red light receptors, phytochromes, at least one blue light receptor, and negative regulatory genes (DET, COP, and FUS) that act downstream from the photoreceptors in the nucleus. In addition, a steroid hormone, brassinolide, also plays a role in light-regulated development and gene expression in Arabidopsis. These molecular and genetic data are allowing us to construct models of the mechanisms by which light controls development and gene expression in Arabidopsis. In the future, this knowledge can be used as a framework for understanding how all land plants respond to changes in their environment.

All metazoan cells are able to make decisions about cell division or cellular differentiation based, in part, on environmental cues. Accordingly, cells express receptor systems that allow them to detect the presence of hormones, growth factors and other signals that manipulate the regulatory processes of the cell. In plants, an unusual signal - light - is required for the induction and regulation of many developmental processes. Past physiological and molecular studies have revealed the variety and complexity of plant responses to light but until recently very little was known about the mechanisms of those responses. Two major breakthroughs have allowed the identification of some photoreceptor signalling intermediates: the identification of photoreceptor and signal transduction mutants inArabidopsis, and the development of single-cell microinjection assays in which outcomes of photoreceptor signalling can be visualized. Here, we review recent genetic advances which support the notion that light responses are not simply endpoints of linear signal transduction pathways, but are the result of the integration of a variety of input signals through a complex network of interacting signalling components.

UNLABELLED Transcription factors and the short, often degenerate DNA sequences they recognize are central regulators of gene expression, but their regulatory code is challenging to dissect experimentally. Thus, computational approaches have long been used to identify putative regulatory elements from the patterns in promoter sequences. Here we present a new algorithm "POWRS" (POsition-sensitive WoRd Set) for identifying regulatory sequence motifs, specifically developed to address two common shortcomings of existing algorithms. First, POWRS uses the position-specific enrichment of regulatory elements near transcription start sites to significantly increase sensitivity, while providing new information about the preferred localization of those elements. Second, POWRS forgoes position weight matrices for a discrete motif representation that appears more resistant to over-generalization. We apply this algorithm to discover sequences related to constitutive, high-level gene expression in the model plant Arabidopsis thaliana, and then experimentally validate the importance of those elements by systematically mutating two endogenous promoters and measuring the effect on gene expression levels. This provides a foundation for future efforts to rationally engineer gene expression in plants, a problem of great importance in developing biotech crop varieties. AVAILABILITY BSD-licensed Python code at http://grassrootsbio.com/papers/powrs/.

Photo synthetic organisms require light to provide the energy necessary to convert CO2 into sugars. Given the non-motile nature of most plants, it is not surprising then that plants have developed systems to alter their growth and developmental patterns in response to the light environment. Indeed, plants exhibit such photomorphogenic responses throughout all stages of the life cycle, from seed germination to floral induction. By studying the wavelength dependence of different responses, two major classes of photoreceptors have been implicated in mediating photomorphogenesis in higher plants: blue/UV photoreceptors and red/far-red photoreceptors. It should be noted at the outset that extensive co-action between these pigment systems has been observed in various species. Until recently, however, only the red/far-red light photoreceptors, called phytochromes, have been identified and characterized at the molecular level.

Oxygenic photosynthetic organisms derive electrons from H2O and evolve molecular oxygen as a by-product of photosynthesis. Such organisms include higher plants, algae, and cyanobacteria. Oxygenic photosynthesis is mediated by two membrane-bound, pigment-protein complexes: Photosystem I (PSI) and Photosystem II (PSII). PSII carries out the reactions resulting in the oxidation of water and the reduction of plastoquinone while PSI utilizes reduced plastoquinone to drive the reduction of NADP. The reaction center of a photosystem is defined as the minimal unit necessary for primary charge separation and stabilization. The solution of the x-ray crystal structure of the anoxygenic bacterial photosynthetic reaction center (Deisenhofer et al., 1985; Allen et al., 1987) led to the prediction (Trebst, 1986) and subsequent verification (Nanba and Satoh, 1987; Marder et al., 1987) that the PSII reaction center consists of a heterodimer of homologous proteins, D1 and D2, and cytochrome b559. The D1/D2 heterodimer is thought to contain all the electron carriers and cofactors necessary for electron transport through the reaction center: P680; the Yz and YD electron donor tyrosines on D1 and D2, respectively; pheophytin; the plastoquinone electron acceptors QA and QB; and non-heme iron (for reviews see Rutherford, 1989; and Mattoo et al., 1989).

Etiolated Avena sativa L. seedlings grown in the presence of gabaculine (5-amino-1,3-cyclohexadienylcarboxylic acid) contained reduced levels of phytochrome as shown by spectrophotometric and immunochemical assays. Photochromic phytochrome levels in gabaculine-grown plants were estimated to be 20% of control plants, while immunoblot analysis showed that the phytochrome protein moiety was present at approximately 50% of control levels. Gabaculine-grown seedlings administered either 5-aminolevulinic acid or biliverdin exhibited a rapid increase of spectrophotometrically detectable phytochrome. Phytochrome concentrations estimated immunochemically did not similarly increase throughout treatment with either compound. Similar experiments with 5-amino[4-14C] levulinic acid showed radiolabeling of phytochrome with kinetics that paralleled the spectrally detected increase. These results are consistent with (a) the intermediacy of both 5-aminolevulinic acid and biliverdin in the biosynthetic pathway of the phytochrome chromophore and (b) the lack of coordinate regulation of chromophore and apoprotein synthesis in Avena seedlings.

A number of photosystem II (PSII) associated proteins, including core proteins D1, D2 and CP43, and several proteins of the LHCII complex, are phosphorylated by a thylakoid-bound, redox-regulated kinase(s). We demonstrate here that the compound propyl gallate is an effective inhibitor of LHCII phosphorylation in vivo while having little effect on PSII core protein phosphorylation. Using this inhibitor, we demonstrate that LHCII dephosphorylation is insensitive to light in vivo. Taken together with our previous conclusion (Elich et al., EMBO J. 12 (1993) 4857–4862) that PSII core protein dephosphorylation is light-stimulated, our data suggest the presence of multiple phosphatases responsible for thylakoid protein dephosphorylation in vivo.

4-Amino-5-hexynoic acid, a suicide inactivator of the mammalian pyridoxal phosphate-dependent 4-aminobutyric acid:2-oxoglutaric acid aminotransferase, inhibits phytochrome and chlorophyll synthesis in developing oat (Avena sativa L.), corn (Zea mays L.), pea (Pisum sativum L.), and cucumber (Cucumis sativus L.) seedlings. In Avena and Cucumis seedlings, respectively, inhibition of phytochrome and chlorophyll accumulation by 4-amino-5-hexynoic acid can be significantly reversed by application of 5-aminolevulinic acid. These results indicate that 4-amino-5-hexynoic acid inhibits the synthesis of 5-aminolevulinic acid in plants.

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.