Tedd D. Elich

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

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.

From lab to field, new approaches to phenotyping root system architecture

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.

Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.

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.

Phytochrome: If It Looks and Smells Like a Histidine Kinase, Is It a Histidine Kinase?

Thats 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.

High-throughput imaging and analysis of root system architecture in Brachypodium distachyon under differential nutrient availability

Nitrogen (N) and phosphorus (P) deficiency are primary constraints for plant productivity, and root system architecture (RSA) plays a vital role in the acquisition of these nutrients. The genetic determinants of RSA are poorly understood, primarily owing to the complexity of crop genomes and the lack of sufficient RSA phenotyping methods. The objective of this study was to characterize the RSA of two Brachypodium distachyon accessions under different nutrient availability. To do so, we used a high-throughput plant growth and imaging platform, and developed software that quantified 19 different RSA traits. We found significant differences in RSA between two Brachypodium accessions grown on nutrient-rich, low-N and low-P conditions. More specifically, one accession maintained axile root growth under low N, while the other accession maintained lateral root growth under low P. These traits resemble the RSA of crops adapted to low-N and -P conditions, respectively. Furthermore, we found that a number of these traits were highly heritable. This work lays the foundation for future identification of important genetic components of RSA traits under nutrient limitation using a mapping population derived from these two accessions.

Initial events in phytochrome signalling: still in the dark

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.

Optimizing root system architecture in biofuel crops for sustainable energy production and soil carbon sequestration.

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.

POWRS: Position-Sensitive Motif Discovery

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/.

Distinct Roles for Intracellular and Extracellular Lipids in Hepatitis C Virus Infection

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.