Convergent evolution of plant prickles by repeated gene co-option over deep time.

An enduring question in evolutionary biology concerns the degree to which episodes of convergent trait evolution depend on the same genetic programs, particularly over long timescales. In this work, we genetically dissected repeated origins and losses of prickles-sharp epidermal projections-that convergently evolved in numerous plant lineages. Mutations in a cytokinin hormone biosynthetic gene caused at least 16 independent losses of prickles in eggplants and wild relatives in the genus .

A Wox3-patterning module organizes planar growth in grass leaves and ligules.

Grass leaves develop from a ring of primordial initial cells within the periphery of the shoot apical meristem, a pool of organogenic stem cells that generates all of the organs of the plant shoot. At maturity, the grass leaf is a flattened, strap-like organ comprising a proximal supportive sheath surrounding the stem and a distal photosynthetic blade. The sheath and blade are partitioned by a hinge-like auricle and the ligule, a fringe of epidermally derived tissue that grows from the adaxial (top) leaf surface.

Protoplast Isolation from Undifferentiated Maize Seedling Shoot Tissue.

Protoplasts are plant cells that have had their cell walls removed, which allows for a variety of cellular manipulations that are not possible within the context of intact plant tissue. Unfortunately, the removal of cell walls is not trivial and can be sensitive to cell type and cell differentiation state. Here, we describe a modified protoplasting protocol that improves isolation of viable protoplasts from the seedling maize shoot apex.

Plant stem-cell organization and differentiation at single-cell resolution.

Plants maintain populations of pluripotent stem cells in shoot apical meristems (SAMs), which continuously produce new aboveground organs. We used single-cell RNA sequencing (scRNA-seq) to achieve an unbiased characterization of the transcriptional landscape of the maize shoot stem-cell niche and its differentiating cellular descendants. Stem cells housed in the SAM tip are engaged in genome integrity maintenance and exhibit a low rate of cell division, consistent with their contributions to germline and somatic cell fates.

Coordination of Leaf Development Across Developmental Axes.

Leaves are initiated as lateral outgrowths from shoot apical meristems throughout the vegetative life of the plant. To achieve proper developmental patterning, cell-type specification and growth must occur in an organized fashion along the proximodistal (base-to-tip), mediolateral (central-to-edge), and adaxial-abaxial (top-bottom) axes of the developing leaf. Early studies of mutants with defects in patterning along multiple leaf axes suggested that patterning must be coordinated across developmental axes.

Plant Development: How Leaves Take Shape.

Live imaging, genetics, and computational modeling reveal how simple versus compound leaves are formed. Cross-species differences in leaf-wide growth determine the outcome of a locally-acting patterning process.

Diverse Sorghum bicolor accessions show marked variation in growth and transcriptional responses to arbuscular mycorrhizal fungi.

Sorghum is an important crop grown worldwide for feed and fibre. Like most plants, it has the capacity to benefit from symbioses with arbuscular mycorrhizal (AM) fungi, and its diverse genotypes likely vary in their responses. Currently, the genetic basis of mycorrhiza-responsiveness is largely unknown.

The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes.

Keeping PASE with WEFT: SHWEFT-PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules.

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from (1)H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion.

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis. A 1H NMR study.

Heme oxygenase (HO), from the pathogenic bacterium N. meningitidis(NmHO), which secures host iron, shares many properties with mammalian HOs but also exhibits some key differences. The crystal structure appears more compact, and the crystal-undetected C-terminus interacts with substrate in solution.

Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH(140-270).

Nine recombinant FixL heme domains from Bradyrhizobium japonicum previously were shown to exhibit mass instability independent of many environmental factors (J.D. Satterlee, C.

The orbital ground state of the azide-substrate complex of human heme oxygenase is an indicator of distal H-bonding: implications for the enzyme mechanism.

The active site electronic structure of the azide complex of substrate-bound human heme oxygenase 1 (hHO) has been investigated by (1)H NMR spectroscopy to shed light on the orbital/spin ground state as an indicator of the unique distal pocket environment of the enzyme. Two-dimensional (1)H NMR assignments of the substrate and substrate-contact residue signals reveal a pattern of substrate methyl contact shifts that places the lone iron pi-spin in the d(xz) orbital, rather than the d(yz) orbital found in the cyanide complex. Comparison of iron spin relaxivity, magnetic anisotropy, and magnetic susceptibilities argues for a low-spin, (d(xy))(2)(d(yz),d(xz))(3), ground state in both azide and cyanide complexes.

Mass instability in isolated recombinant FixL heme domains of Bradyrhizobium japonicum.

Several recombinant Bradyrhizobium japonicum FixL heme domains (BjFixLH) have been characterized and their temporal mass stabilities assessed by MALDI-TOF mass spectrometry. The intact heme domains all bound heme and gave normal UV-visible spectra, indicating that they were correctly assembled. Proteins produced at Washington State University included a parent 131-amino acid "full-length heme domain" (FLHD) of primary sequence T140-Q270 (BjFixLH140-270), a histidine-tagged analogue containing an N-terminal extension, and five different terminus-truncated variants.

Evidence for fast conformational change upon ligand dissociation in the HemAT class of bacterial oxygen sensors.

Here we report the results of transient absorption and photoacoustic calorimetry studies of CO photodissociation from the heme domain of the bacterial oxygen sensor HemAT-Bs. The results indicate that CO photolysis is accompanied by an overall DeltaH of -19 kcal mol(-1) and DeltaV of +4 ml mol(-1) as well as a red-shifted kinetic difference spectrum all occurring in <50 ns. Analysis of the DeltaH/DeltaV reveals that a conformational change takes place with a DeltaH(conf) of -40 kcal mol(-1) and DeltaV(conf) of -22 ml mol(-1).

Small molecule directed aggregation of a heme peptide on gold: an STM study.

Microperoxidase 11 was adsorbed on Au(111) from basic aqueous solutions containing pure heme peptide and co-added stoichiometric amounts of exogenous neutral and ionic ligands. The addition of small molecules to MP11 produced different aggregate structures that were easily differentiated by STM. In the absence of a complexing agent, the MP-11 formed large clusters of metallopeptide molecules on the gold surface.

1H NMR Study of the influence of hemin vinyl-->methyl substitution on the interaction between the C-terminus and substrate and the "aging" of the heme oxygenase from Neisseria meningitidis: induction of active site structural heterogeneity by a two-fold symmetric hemin.

Solution 1H NMR has been used to characterize the active site molecular and electronic structure of the cyanide-inhibited 2,4-dimethyldeuterohemin complex of the heme oxygenase from Neisseria meningitidis (NmHO) with respect to the mode of interaction of the C-terminus with the substrate and the spontaneous "aging" of NmHO that results in the cleavage of the C-terminal Arg208-His209 dipeptide. The structure of the portion involving residues Ala12-Phe192 is found to be essentially identical to that of the protohemin complex in either solution or crystal. However, His207 from the C-terminus is found to interact strongly with the substrate 1CH3, as opposed to the 8CH3 in the protohemin complex.

Characterization of the spontaneous "aging" of the heme oxygenase from the pathological bacterium Neisseria meningitidis via cleavage of the C-terminus in contact with the substrate. Implications for functional studies and the crystal structure.

Solution 1H NMR spectroscopy and mass spectrometry are utilized to characterize the irreversible "aging" of native heme oxygenase from N. meningitidis, NmHO. 2D NMR characterization of the cyanide-inhibited substrate complex shows that the C-terminal interaction between Arg208His209 and the exposed pyrrole of the protohemin substrate in the "native" NmHO complex is lost in the "aging".

Characterization of conformational changes coupled to ligand photodissociation from the heme binding domain of FixL.

Using transient absorption spectroscopy and photoacoustic calorimetry (PAC), we have characterized carbon monoxide photodissociation and rebinding to two forms of the heme domain of Bradyrhizobium japonicum FixL. Transient absorption results for the complete heme domain (FixL residues 140-270) and a truncated heme domain (missing 11 residues on the N-teminal end and 14 amino acid residues on the C-terminal end of the full length heme domain) show similar rates for ligand rebinding to the five-coordinate heme domain and the absence of any transient intermediate on a microsecond time scale. Results from PAC studies show that both the truncated and complete heme domains undergo a contraction upon ligand photolysis.

Recombinant PAS-heme domains of oxygen sensing proteins: high level production and physical characterization.

Details of a high-level recombinant production method for the heme-PAS domains of heme oxygen sensing proteins from Sinorhizobium meliloti (Sm) (formerly Rhizobium meliloti, Rm), Bradyrhizobium japonicum (Bj), and Escherichia coli (Ec) are described. Using a newly proposed, concise, and unambiguous naming system (also described here) these proteins are: SmFixLH(128-264), BjFixLH(140-270), and EcDosH(1-147). In addition, high-level production of BjFixL(140-505), the soluble full-length protein containing both heme (oxygen sensing) and kinase (catalytic) domains is described.

Insights into signal transduction involving PAS domain oxygen-sensing heme proteins from the X-ray crystal structure of Escherichia coli Dos heme domain (Ec DosH).

The X-ray crystal structure of the Escherichia coli (Ec) direct oxygen sensor heme domain (Ec DosH) has been solved to 1.8 A using Fe multiple-wavelength anomalous dispersion (MAD), and the positions of Met95 have been confirmed by selenomethionine ((Se)Met) MAD. Ec DosH is the sensing part of a larger two-domain sensing/signaling protein, in which the signaling domain has phosphodiesterase activity.

A superoxo-ferrous state in a reduced oxy-ferrous hemoprotein and model compounds.

Cryoreduction of the [FeO2]6 (n = 6 is the number of electrons in 3d orbitals on Fe and pi* orbitals on O2) dioxygen-bound ferroheme through irradiation at 77 K generates an [FeO2]7 reduced oxy-heme. Numerous investigations have examined [FeO2]7 centers that have been characterized as peroxo-ferric centers, denoted [FeO2]per7, in which a ferriheme binds a dianionic peroxo-ligand. The generation of such an intermediate can be understood heuristically if the [FeO2]6 parent is viewed as a superoxo-ferric center and the injected electron localizes on the O-O moiety.

Temperature, pH, and solvent isotope dependent properties of the active sites of resting-state and cyanide-ligated recombinant cytochrome c peroxidase (H52L) revealed by proton hyperfine resonance spectra.

Comparative proton NMR studies have been carried out on high-spin and low-spin forms of recombinant native cytochrome c peroxidase (rCcP) and its His52 --> Leu variant. Proton NMR spectra of rCcP(H52L) (high spin) and rCcP(H52L)CN (low spin) reveal the presence of multiple enzyme forms in solution, whereas only single enzyme forms are found in spectra of wild-type and recombinant wild-type CcP and CcPCN near neutral pH. The spectroscopic behaviors of these forms have been studied in detail when pH, temperature, and solvent isotope composition were varied.

Cyanide binding to cytochrome c peroxidase (H52L).

Cyanide binding to a cytochrome c peroxidase (CcP) variant in which the distal histidine has been replaced by a leucine residue, CcP(H52L), has been investigated as a function of pH using spectroscopic, equilibrium, and kinetic methods. Between pH 4 and 8, the apparent equilibrium dissociation constant for the CcP(H52L)/cyanide complex varies by a factor of 60, from 135 microM at pH 4.7 to 2.

Crystal structures of unligated and CN-ligated Glycera dibranchiata monomer ferric hemoglobin components III and IV.

Erythrocytes of the marine annelid, Glycera dibranchiata, contain a mixture of monomeric and polymeric hemoglobins. There are three major monomer hemoglobin components, II, III, IV (also called GMH2, 3, and 4), that have been highly purified and well characterized. We have now crystallized GMH3 and GMH4 and determined their structures to 1.

Cloning, purification, crystallization and preliminary X-ray analysis of DOS heme domain, a new heme oxygen sensor in Escherichia coli.

The heme-containing PAS domain of the direct oxygen-sensor protein (DOS(H)), a bona fide oxygen-sensor protein, has been cloned from Escherichia coli strain K12 and successfully purified. The oxidized form of this protein was crystallized by the hanging-drop method with a PEG 8000-based precipitant. Preliminary X-ray diffraction studies of the PAS-domain crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.