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. Surprisingly, we find no evidence for a canonical stem-cell organizing center subtending these cells. In addition, trajectory inference was used to trace the gene expression changes that accompany cell differentiation, revealing that ectopic expression of KNOTTED1 (KN1) accelerates cell differentiation and promotes development of the sheathing maize leaf base. These single-cell transcriptomic analyses of the shoot apex yield insight into the processes of stem-cell function and cell-fate acquisition in the maize seedling and provide a valuable scaffold on which to better dissect the genetic control of plant shoot morphogenesis at the cellular level.

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. Here, we investigated transcriptional and physiological responses of sorghum accessions, founders of a bioenergy nested association mapping panel, for their responses to four species of AM fungi. Transcriptome comparisons across four accessions identified mycorrhiza-inducible genes; stringent filtering criteria revealed 278 genes that show mycorrhiza-inducible expression independent of genotype and 55 genes whose expression varies with genotype. The latter suggests variation in phosphate transport and defence across these accessions. The mycorrhiza growth and nutrient responses of 18 sorghum accessions varied tremendously, ranging from mycorrhiza-dependent to negatively mycorrhiza-responsive. Additionally, accessions varied in the number of AM fungi to which they showed positive responses, from one to several fungal species. Mycorrhiza growth and phosphorus responses were positively correlated, whereas expression of two mycorrhiza-inducible phosphate transporters, SbPT8 and SbPT9, correlated negatively with mycorrhizal growth responses. AM fungi improve growth and mineral nutrition of sorghum, and the substantial variation between lines provides the potential to map loci influencing mycorrhiza responses.

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. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

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 Solanum. Homologs underlie prickle formation across angiosperms that collectively diverged more than 150 million years ago, including rice and roses. By developing new Solanum genetic systems, we leveraged this discovery to eliminate prickles in a wild species and an indigenously foraged berry. Our findings implicate a shared hormone activation genetic program underlying evolutionarily widespread and recurrent instances of plant morphological innovation.

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. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform-paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple (1)H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co(2+)-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH(151-256), the heme sensing domain of Bradyrhizobium japonicum FixL.

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. Together, the ligule and auricle comprise morphological novelties that are specific to grass leaves. Understanding how the planar outgrowth of grass leaves and their adjoining ligules is genetically controlled can yield insight into their evolutionary origins. Here we use single-cell RNA-sequencing analyses to identify a 'rim' cell type present at the margins of maize leaf primordia. Cells in the leaf rim have a distinctive identity and share transcriptional signatures with proliferating ligule cells, suggesting that a shared developmental genetic programme patterns both leaves and ligules. Moreover, we show that rim function is regulated by genetically redundant Wuschel-like homeobox3 (WOX3) transcription factors. Higher-order mutations in maize Wox3 genes greatly reduce leaf width and disrupt ligule outgrowth and patterning. Together, these findings illustrate the generalizable use of a rim domain during planar growth of maize leaves and ligules, and suggest a parsimonious model for the homology of the grass ligule as a distal extension of the leaf sheath margin.

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.

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. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D (1)H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross-peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin, and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~10(2) increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed.

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. The switch from singly occupied d(yz) for the cyanide to d(xz) for the azide complex of hHO is shown to be consistent with the orbital hole determined by the azide pi-plane in the latter complex, which is approximately 90 degrees in-plane rotated from that of the imidazole pi-plane. The induction of the altered orbital ground state in the azide relative to the cyanide hHO complex, as well as the mean low-field bias of methyl hyperfine shifts and their paramagnetic relaxivity relative to those in globins, indicates that azide exerts a stronger ligand field in hHO than in the globins, or that the distal H-bonding to azide is weaker in hHO than in globins. The Asp140 --> Ala hHO mutant that abolishes activity retains the unusual WT azide complex spin/orbital ground state. The relevance of our findings for other HO complexes and the HO mechanism is discussed.

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. Decades later, we now recognize that a highly complex and interconnected transcriptional network of patterning genes and hormones underlies leaf development. Here, we review the molecular genetic mechanisms by which leaf development is coordinated across leaf axes. Such coordination likely plays an important role in ensuring the reproducible phenotypic outcomes of leaf morphogenesis.

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.

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). These thermodynamic changes are consistent with an increase in the solvent accessible surface area of the protein upon ligand dissociation, as observed in the X-ray structure of the ferric CN-bound and CN free forms of HemAT-Bs.

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. With neutral imidazole in solution the MP11 aggregated into regular elongated structures (nano-épis) on the substrate. When S(2-) is used as coupling agent, single heme peptide molecules are isolated with identifiable porphyrin ring and substructure in the peptide chain.

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 1.4-1.8 A resolution. The structures were determined for these two monomer hemoglobins in the oxidized (Fe3+, ferric, or met-) forms in both the unligated and cyanide-ligated states. This work differs from two published, refined structures of a Glycera dibranchiata monomer hemoglobin, which has a sequence that is substantially different from any bona fide major monomer hemoglobins (GMH2, 3, or 4). The high-resolution crystal structures (presented here) and the previous NMR structure of CO-ligated GMH4, provide a basis for interpreting structure/function details of the monomer hemoglobins. These details include: (1) the strong correlation between temperature factor and NMR dynamics for respective protein forms; (2) the unique nature of the HisE7Leu primary sequence substitutions in GMH3 and GMH4 and their impact on cyanide ion binding kinetics; (3) the LeuB10Phe difference between GMH3 and GMH4 and its impact on ligand binding; and (4) elucidation of changes in the structural details of the distal and proximal heme pockets upon cyanide binding.

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. Suquet, A. Bidwai, J. Erman, L. Schwall, R. Jimenez, Biochemistry 47 (2008) 1540-1553). Two of those recombinant proteins were produced in remote laboratories. Mass losses begin appearing at completion of isolation and comprise a substantial proportion of samples within 1-3 days of storage and handling. Thus, degradation occurs during the time frame of experiments and crystallization. Detailed understanding of this instability is desired in order to formulate stable heme-PAS sensor domains for experimentation and for a mechanistic interpretation. However, mass spectra of the full length heme-PAS domain, BjFixLH(140-270), are complex by 1-3 days following isolation due to broad features and a high density of overlapping peaks, so that individual peak assignments are at present ambiguous. This stymies direct, quantitative interpretation of the source of the observed mass losses. To solve this dilemma amino-terminal primary sequencing and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry monitoring of three terminal variants of BjFixLH(140-270) have been achieved. The working hypothesis, that the experimentally observed mass losses originate in the PAS protein sequence termini, has been substantiated. This establishes a basis for interpreting the more complex results from aging full length BjFixLH(140-270).

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. The smallest of these was a 106-amino acid "core PAS heme domain" with primary sequence T151-L256. All variants except for the smallest exhibited significant mass instability, assessed by MALDI-TOF mass spectrometry, that was apparent within 1-16 days standing in a sterile environment at room temperature. Two full-length heme domains expressed independently in geographically remote laboratories (Northern Illinois University and JILA, University of Colorado) also exhibited this mass instability. A mass loss of as much as approximately 25% of the starting mass has been observed, which could explain the "missing" terminal amino acids in published crystal structures. This work documents the phenomenon and its persistence despite (i) sample sterilization, (ii) protease inhibitors, (iii) primary sequence variations, (iv) the presence or absence of ferriheme ligands, and (v) the presence or absence of O2.

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". Mass spectrometry and N-terminal sequencing of wild type and "aged" NmHO reveal that the "aging" process involves cleavage of the Arg208His209 dipeptide. The construction of the double deletion mutant without Arg208His209 and its NMR comparison as both the resting state substrate complex and its cyanide-inhibited complex with the "aged" NmHO reveal that cleavage of the C-terminal dipeptide is the only modification during the aging. Comparison of cyanide ligand binding constants reveal a factor approximately 1.7 greater CN- affinity in the native than "aged" NmHO. The rate of protohemin degradation and its stereoselectivity are unaffected by the C-terminal truncation. However, the free alpha-biliverdin yield in the presence of desferrioxamine is significantly increased in the "aged" NmHO and its deletion mutant relative to WT, arguing for a role of the NmHO C-terminus in modulating product release. The facile cleavage of Arg208His209 in the resting state complex, with a half-life of approximately 24 h at 25 degrees C, suggests that previous characterization of NmHO may have been carried out on a mixture of native and "aged" NmHO, and may account for the "lost" C-terminal residues in the crystal structures.

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. The different mode of interaction of His207 with the alternate substrates is attributed to the 2-vinyl group of protohemin sterically interfering with the optimal orientation of the proximal helix Asp27 carboxylate that serves as acceptor to the strong H-bond by the peptide of His207. The 2,4-dimethyldeuterohemin HO complex "ages" in manner similary to that of protohemin, (Liu, Y., Ma, L.-H., Satterlee, J.D., Zhang, X., Yoshida, T., and La Mar, G. N., (2006) Biochemistry 45, 3875-3886) with mass spectrometry and N-terminal sequencing indicating that the Arg208-His209 dipeptide is cleaved. The 2,4-dimethyldeuterohemin complex of WT HO populates an equilibrium isomer stabilized in low phosphate concentration for which the axial His imidazole ring is rotated by approximately 20 degrees from that in the WT. The His ring reorientation is attributed to Asp24 serving as the H-bond acceptor to the His207 peptide NH, rather than to the His23 ring NdeltaH as in the crystals. The functional implications of the altered C-terminal interaction with substrate modification are discussed.

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. Using an IPTG-inducible pET/BL21 expression system and a rapid, two-column purification has resulted in increased yields of 3- to 17-fold over literature values. The recombinant proteins are highly pure as judged by SDS-PAGE, MALDI-TOF mass spectrometry, and a UV-visible purity index. To our knowledge, this work includes the first mass spectrometry analysis of any PAS-heme protein and provides high-resolution confirmation of each protein's identity. These production and characterization improvements make possible future spectroscopic and dynamics studies designed to elucidate the intramolecular/interdomain signal that follows heme-domain oxygen dissociation.

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. In addition, CO photolysis from the complete heme domain gives rise to an intermediate with a lifetime of approximately 150 ns which is absent in the truncated heme domain. We attribute the 150 ns phase to ligand release to the solvent which may be accelerated in the case of the truncated domain. The initial contraction is attributed to changes in the charge distribution due to reorganization of the surface salt bridge formed between Glu182 and Arg227 or possibly to reorientation of Arg206. Changes in the charge distribution may play an important role in communication between the sensor domain and the regulatory domain and thus may be part of the signal transduction pathway.