Thomas Schultz

ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis

We have conducted genetic screens for period length mutants in Arabidopsis using a transgenic bioluminescence phenotype. This screen identified mutations at a locus, ZEITLUPE (ZTL), that lengthen the free-running period of clock-controlled gene transcription and cell expansion, and alter the timing of the daylength-dependent transition from vegetative to floral development. Map-based cloning of ZTL identified a novel 609 amino acid polypeptide consisting of an amino-terminal PAS domain, an F box and six carboxy-terminal kelch repeats. The PAS region is highly similar to the PAS domain of the Arabidopsis blue-light receptor NPH1, and the Neurospora circadian-associated protein WHITE COLLAR-1 (WC-1). The striking fluence rate-dependent effect of the ztl mutations suggests that ZTL plays a primary role in the photocontrol of circadian period in higher plants.

The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana. Here we identify a protein complex (called the evening complex)—composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)—that directly regulates plant growth. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4–ELF3–LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

Efficient deactivation of a model base pair via excited-state hydrogen transfer

We present experimental and theoretical evidence for an excited-state deactivation mechanism specific to hydrogen-bonded aromatic dimers, which may account, in part, for the photostability of the Watson-Crick base pairs in DNA. Femtosecond time-resolved mass spectroscopy of 2-aminopyridine clusters reveals an excited-state lifetime of 65 ± 10 picoseconds for the near-planar hydrogen-bonded dimer, which is significantly shorter than the lifetime of either the monomer or the 3- and 4-membered nonplanar clusters. Ab initio calculations of reaction pathways and potential-energy profiles identify the mechanism of the enhanced excited-state decay of the dimer: Conical intersections connect the locally excited 1ππ* state and the electronic ground state with a 1ππ* charge-transfer state that is strongly stabilized by the transfer of a proton.

14-3-3 Proteins Are Part of an Abscisic Acid-VIVIPAROUS1 (VP1) Response Complex in the Em Promoter and Interact with VP1 and EmBP1

Protein–DNA complexes were formed when nuclear extracts from embryogenic rice suspension cultures or maize embryos were incubated with an abscisic acid–VIVIPAROUS1 (VP1) response element (Em1a) from the Em promoter. Monoclonal antibodies generated to GF14, a 14-3-3 protein from plants, resulted in gel retardation of the Em1a–protein complexes. Antibodies generated to the C and N termini of GF14 detected protein isoforms in rice nuclear and cytoplasmic extracts, but no differences in distribution of the GF14 isoforms were recognized between the nucleus and cytoplasm or when abscisic acid–treated and untreated tissues were compared. When recombinant GF14 fusion proteins from rice were added to nuclear extracts, novel complexes were formed that required the dimerization domain of GF14. Chemical cross-linking showed that GF-14 interacted with the basic leucine zipper factor EmBP1, which binds specifically to Em1a, and with VP1, which transactivates Em through Em1a. GF14 proteins from rice were shown to interact with VP1 in yeast through the dimerization domain of GF14. Our results indicated that GF14 interacts with both site-specific DNA binding proteins (i.e., EmBP1) and tissue-specific regulatory factors (i.e., VP1) and may provide a structural link between VP1 and the Em1a transcriptional complex.

Diatom gliding is the result of an actin-myosin motility system

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.

Rapid Array Mapping of Circadian Clock and Developmental Mutations in Arabidopsis

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F2 individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.

Excited-state dynamics of cytosine tautomers.

We report the relaxation dynamics of keto and enol or keto-imino cytosine, photoexcited in the wavelength range of 260-290 nm. Three transients with femtosecond to hundreds of picoseconds lifetimes are observed for the biologically relevant keto tautomer and are assigned to internal conversion and excited-state tautomerization. Only two transients with femtosecond and picosecond lifetimes are identified for the enol or keto-imino tautomer and are assigned to internal conversion processes. The results are discussed in the context of published ab initio theory.

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.

Relevance of πσ* states in the photoinduced processes of adenine, adenine dimer, and adenine-water complexes

Ab initio calculations and time-resolved photoionization spectroscopy were carried out to characterize the role of the lowest two πσ* excited states for the photoinduced processes in the adenine monomer, adenine dimer, and adenine–water clusters. The calculations show—with respect to the monomer—a stabilization of 0.11–0.14 eV for the πσ* states in different isomers of adenine dimer and an even bigger stabilization of 0.14–0.36 eV for isomers of adenine-(H2O)1 and adenine-(H2O)3. Hence, the stabilized πσ* states should play an important role in the excited-state relaxation of partially or fully solvated adenine. This conclusion is supported by experimental results: In the adenine monomer, strong nπ* state signals are observed. Those signals are reduced in adenine dimer and vanish in water clusters due to the competing relaxation via the πσ* states.

DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species‐specific, semi‐quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10‐fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.