IscR (iron-sulfur cluster regulator) is encoded by an ORF located immediately upstream of genes coding for the Escherichia coli Fe-S cluster assembly proteins, IscS, IscU, and IscA. IscR shares amino acid similarity with MarA, a member of the MarA͞SoxS͞Rob family of transcription factors. In this study, we found that IscR functions as a repressor of the iscRSUA operon, because strains deleted for iscR have increased expression of this operon. In addition, in vitro transcription reactions established a direct role for IscR in repression of the iscR promoter. Analysis of IscR by electron paramagnetic resonance showed that the anaerobically isolated protein contains a [2Fe-2S] 1؉ cluster. The Fe-S cluster appears to be important for IscR function, because repression of iscR expression is significantly reduced in strains containing null mutations of the Fe-S cluster assembly genes iscS or hscA. The finding that IscR activity is decreased in strain backgrounds in which Fe-S cluster assembly is impaired suggests that this protein may be part of a novel autoregulatory mechanism that senses the Fe-S cluster assembly status of cells. to toxic levels. An important finding in recent years is the discovery of a conserved set of proteins in organisms from bacteria to humans that facilitate assembly of Fe-S clusters into Fe-S proteins (1, 2). In several bacteria, the genes encoding these Fe-S assembly proteins (IscS, IscU, IscA, Hsc66, Hsc20, and ferredoxin) are organized in a cluster, iscSUAhscBAfdx. In this study, we have focused on the regulation of the expression of the genes that encode the Fe-S cluster assembly factors from Escherichia coli.Genetic experiments support the key role of the proteins encoded by iscSUAhscBAfdx in Fe-S cluster assembly, because mutations in the E. coli genes decrease the activity of many Fe-S proteins (3-5). Furthermore, biochemical studies have begun to provide some insight into the process of Fe-S cluster assembly (1, 6-10). IscS is a cysteine desulfurase that procures the sulfur from cysteine for Fe-S cluster assembly (1, 7). IscU can form a complex with IscS and acquire an unstable [2Fe-2S] cluster that has been proposed to be a source of Fe and sulfur for Fe-S protein assembly (6,8,11). IscA can also acquire a Fe-S cluster in vitro (9). Hsc66 and Hsc20, homologs to molecular chaperones, form a complex in vitro with IscU, but the exact physiological function of this interaction has yet to be determined (10).It is also of interest to understand how expression of the Fe-S cluster assembly proteins is controlled. In bacteria, the cellular requirements for Fe-S cluster assembly must vary, because the synthesis of many Fe-S proteins is regulated (12) and environmental conditions arise that lead to the destruction of these metal centers (13). Thus, it would not be surprising to find that expression of the assembly proteins is regulated to accommodate such changes in Fe-S cluster assembly requirements. A clue that the genes encoding Fe-S cluster assembly proteins might be regulated came from the...

Plants can defend themselves against a wide array of enemies, yet one of the most striking observations is the variability in the effectiveness of such defences, both within and between species. Some of this variation can be explained by conflicting pressures from pathogens with different modes of attack1. A second explanation comes from an evolutionary tug of war, in which pathogens adapt to evade detection, until the plant has evolved new recognition capabilities for pathogen invasion2-5. If selection is, however, sufficiently strong, susceptible hosts should remain rare. That this is not the case is best justified by costs incurred from constitutive defences in a pest free environment6-11. Using a combination of forward genetics and genome-wide association analyses, we demonstrate that allelic diversity at a single locus, ACCELERATED CELL DEATH 6 (ACD6)12,13, underpins dramatic pleiotropic differences in both vegetative growth and resistance to microbial infection and herbivory among natural Arabidopsis thaliana strains. A hyperactive ACD6 allele, compared to the reference allele, strongly enhances resistance to a broad range of pathogens from different phyla, but at the same time slows the production of new leaves and greatly reduces the biomass of mature leaves. This allele segregates at intermediate frequency both throughout the worldwide range of A. thaliana and within local populations, consistent with this allele providing substantial fitness benefits despite its drastic impact on growth.

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization is an example of temperature influencing the timing of flowering and is defined as the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring. In cereals, three genes (VERNALIZATION1 [VRN1], VRN2, and FLOWERING LOCUS T [FT]) have been identified that influence the vernalization requirement and are thought to form a regulatory loop to control the timing of flowering. Here, we characterize natural variation in the vernalization and photoperiod responses in Brachypodium distachyon, a small temperate grass related to wheat (Triticum aestivum) and barley (Hordeum vulgare). Brachypodium spp. accessions display a wide range of flowering responses to different photoperiods and lengths of vernalization. In addition, we characterize the expression patterns of the closest homologs of VRN1, VRN2 (VRN2-like [BdVRN2L]), and FT before, during, and after cold exposure as well as in different photoperiods. FT messenger RNA levels generally correlate with flowering time among accessions grown in different photoperiods, and FT is more highly expressed in vernalized plants after cold. VRN1 is induced by cold in leaves and remains high following vernalization. Plants overexpressing VRN1 or FT flower rapidly in the absence of vernalization, and plants overexpressing VRN1 exhibit lower BdVRN2L levels. Interestingly, BdVRN2L is induced during cold, which is a difference in the behavior of BdVRN2L compared with wheat VRN2 during cold.

The cysteine desulfurase, IscS, provides sulfur for Fe-S cluster synthesis in vitro, but a role for IscS in in vivo Fe-S cluster formation has yet to be established. To study the in vivo function of IscS in Escherichia coli, a strain lacking IscS was constructed and characterized. Using this iscS deletion strain, we have observed decreased specific activities for proteins containing [4Fe-4S] clusters from soluble (aconitase B, 6-phosphogluconate dehydratase, glutamate synthase, fumarase A, and FNR) and membrane-bound proteins (NADH dehydrogenase I and succinate dehydrogenase). A specific role for IscS in in vivo Fe-S cluster assembly was demonstrated by showing that an Fe-S cluster independent mutant of FNR is unaffected by the lack of IscS. These data support the conclusion that, via its cysteine desulfurase activity, IscS provides the sulfur that subsequently becomes incorporated during in vivo Fe-S cluster synthesis. We also have characterized a growth phenotype associated with the loss of IscS. Under aerobic conditions the deletion of IscS caused an auxotrophy for thiamine and nicotinic acid, whereas under anaerobic conditions, only nicotinic acid was required. The lack of IscS also had a general effect on the growth of E. coli because the iscS deletion strain grew at half the rate of wild type in many types of media even when the auxotrophies were satisfied.NifS ͉ FNR ͉ oxidative stress repair ͉ sulfur metabolism

Plants of the Cannabis genus are the only prolific producers of phytocannabinoids, compounds that strongly interact with the evolutionarily ancient endocannabinoid receptors shared by most bilaterian taxa. For millennia, the plant has been cultivated not only for these compounds, but also for food, rope, paper, and clothing. Today, specialized varieties yielding high-quality textile fibers, nutritional seed oil, or high cannabinoid content are cultivated across the globe. However, the genetic identities and histories of these diverse populations remain largely obscured. We analyzed the nuclear genomic diversity among 340 Cannabis varieties, including fiber and seed oil hemp, high cannabinoid drug-types, and feral populations. These analyses demonstrate the existence of at least three major groups of diversity with European hemp varieties more closely related to narrow leaflet drug-types (NLDTs) than to broad leaflet drug-types (BLDTs). The BLDT group appears to encompass less diversity than the NLDT, which reflects the larger geographic range of NLDTs, and suggests a more recent origin of domestication of the BLDTs. As well as being genetically distinct, hemp, NLDT, and BLDT genetic groups produce unique cannabinoid and terpenoid content profiles. This combined analysis of population genomic and trait variation informs our understanding of the potential uses of different genetic variants for medicine and agriculture, providing valuable insights and tools for a rapidly emerging valuable industry.

Cannabis has been cultivated for millennia with distinct cultivars providing either fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than 20 tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol/cannabidiol segregating population and a complete chromosome assembly from a high-cannabidiol cultivar together resolve the linkage 25 of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated with transposable elements. High-cannabidiol cultivars appear to have been generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests that overall drug potency, however, is associated with other genomic regions needing 30 additional study. Resources available online at: http://cannabisgenome.org SummaryA complete chromosome assembly and an ultra-high-density linkage map together identify the genetic mechanism responsible for the ratio of tetrahydrocannabinol 35 (THC) to cannabidiol (CBD) in Cannabis cultivars, allowing paradigms for the evolution and inheritance of drug potency to be evaluated. 45 has also frustrated attempts to assemble complete chromosomes from thousands of Veronica Tonnell contributed DNA isolation for the mapping population. We thank

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (F T), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.

Summary Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore‐based assembly anchored to a high‐resolution linkage map provided a chromosome‐resolved genome for CBDRx, a potent CBD‐type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed. Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD‐type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio. Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.