Summary Light plays a profound role in plant development, yet how photoreceptor excitation directs phenotypic plasticity remains elusive. One of the earliest effects of light is the regulated translocation of the red/far-red photoreceptors, phytochromes, from the cytoplasm to subnuclear foci called phytochrome nuclear bodies. The function of these nuclear bodies is unknown. We report the identification of hemera, a seedling lethal mutant of Arabidopsis with altered phytochrome nuclear body patterns. hemera mutants are impaired in all phytochrome responses examined, including proteolysis of phytochrome A and phytochrome-interacting transcription factors. HEMERA was identified previously as pTAC12, a component of a plastid complex associated with transcription. Here we show that HEMERA has a function in the nucleus, where it acts specifically in phytochrome signaling, is predicted to be structurally similar to the multiubiquitin-binding protein, RAD23, and can partially rescue yeast rad23mutants. Together, these results implicate phytochrome nuclear bodies as sites of proteolysis.

Growth in plants is modulated by a complex interplay between internal signals and external cues. Although traditional mutagenesis has been a successful approach for the identification of growth regulatory genes, it is likely that many genes involved in growth control remain to be discovered. In this study, we used the phenotypic variation between Bay-0 and Shahdara, two natural strains (accessions) of Arabidopsis thaliana, to map quantitative trait loci (QTL) affecting light-and temperature-regulated growth of the embryonic stem (hypocotyl). Using heterogeneous inbred families (HIFs), the gene underlying one QTL, LIGHT5, was identified as a tandem zinc knuckle/PLU3 domain encoding gene (At5g43630; TZP), which carries a premature stop codon in Bay-0. Hypocotyl growth assays in monochromatic light and microarray analysis demonstrate that TZP controls blue light associated growth in a time-of-day fashion by regulating genes involved in growth, such as peroxidase and cell wall synthesis genes. TZP expression is phased by the circadian clock and light/dark cycles to the beginning of the day, the time of maximal growth in A. thaliana in short-day conditions. Based on its domain structure and localization in the nucleus, we propose that TZP acts downstream of the circadian clock and photoreceptor signaling pathways to directly control genes responsible for growth. The identification of TZP thus provides new insight into how daily synchronization of growth pathways plays a critical role in growth regulation.blue light ͉ circadian ͉ fine-mapping ͉ quantitative T he embryonic stem or hypocotyl is an excellent model for studying both internal and external factors controlling growth in plants (1). Genetic screens in common laboratory accessions have yielded direct molecular insight into how light-and hormonedependent signaling pathways interact with the circadian clock to regulate the final length of the hypocotyl (1). The power of the hypocotyl assay is its simplicity, as well as its obvious meaningfulness. When germinating seeds are exposed to low levels of light, such as those caused by a covering layer of debris, the hypocotyl has to grow for a while. Only after the surface has been broken by the tip of the hypocotyls can the embryonic leaves, the cotyledons, unfold. Conversely, if a seed has fallen on open ground, there is no need for the hypocotyls to be particularly long. Because of the ease and reproducibility with which hypocotyl length can be measured in thousands of individuals, it has also been a powerful model in mapping genes with more subtle effects on light and hormone regulated growth, by using methods of quantitative genetics (2). Multiple light signaling genes controlling hypocotyl length have been characterized in quantitative trait locus (QTL) studies (3-6).In this study, we use the hypocotyl assay to identify QTL controlling growth in 2 light and 2 temperature conditions. We identified a recessive large effect QTL on chromosome five controlling 40% of the growth variation segregating in Recombinant Inb...

Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in R. sphaeroides, and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival.

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.

While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris. IMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

The ability to simultaneously and more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms yet has thus far been challenging. Here, we describe a method to help address this problem, which we dub “Met-Seq” (metabolite-coupled Tn sequencing).

18While lignin represents a major fraction of the carbon in plant biomass, biological strategies to 19 convert the components of this heterogenous polymer into products of industrial and 20 biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a 21 byproduct of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin 22 streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known 23 degrader of phenolic compounds under photoheterotrophic conditions, via the benzoyl-CoA 24 degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to 25 metabolize meta-methoxylated phenolics such as syringic acid. We isolated a strain of R. palustris 26 (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under 27 photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. 28 An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow 29 on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the 30 BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement 31 of products of the vanARB operon (rpa3619-rpa3621), which has been described as catalyzing 32 aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In 33 addition, experiments with a vanARB deletion mutant demonstrated the involvement of the 34 vanARB operon in anaerobic syringic acid degradation. These observations provide new insights 35 into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris. 36 IMPORTANCE 37Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually 38 substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological 39 applications. Engineering microorganisms for production of aromatic-based biochemicals requires 40 3 detailed knowledge of metabolic pathways for the degradation of aromatics that are present in 41 lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid 42 degradation reveals a previously unknown metabolic route for aromatic degradation in R. palustris. 43 This study highlights several key features of this pathway and sets the stage for a more complete 44 understanding of the microbial metabolic repertoire to metabolize aromatic compounds from lignin 45 and other renewable sources. 46 48 renewable source of carbon for the bio-based production of compounds that are currently derived 49 from petroleum. Unfortunately, the ability to derive chemicals of commercial, chemical, or 50 medicinal value from lignin is limited by information needed to improve the biological conversion 51 of the aromatics in lignin into valuable products. We are interested in improving our understanding 52 of how bacteria metabolize the aromatic building blocks in lignin and using this information to 53 devel...