Debra M. Sherman

Actin Purified from Maize Pollen Functions in Living Plant Cells.

A vast array of actin binding proteins (ABPs), together with intracellular signaling molecules, modulates the spatiotemporal distribution of actin filaments in eukaryotic cells. To investigate the complex regulation of actin organization in plant cells, we designed experiments to reconstitute actin-ABP interactions in vitro with purified components. Because vertebrate skeletal [alpha]-actin has distinct and unpredictable binding affinity for nonvertebrate ABPs, it is essential that these in vitro studies be performed with purified plant actin. Here, we report the development of a new method for isolating functional actin from maize pollen. The addition of large amounts of recombinant profilin to pollen extracts facilitated the depolymerization of actin filaments and the formation of a profilin-actin complex. The profilin-actin complex was then isolated by affinity chromatography on poly-L-proline-Sepharose, and actin was selectively eluted with a salt wash. Pollen actin was further purified by one cycle of polymerization and depolymerization. The recovery of functional actin by this rapid and convenient procedure was substantial; the average yield was 6 mg of actin from 10 g of pollen. We undertook an initial physicochemical characterization of this native pollen actin. Under physiological conditions, pollen actin polymerized with kinetics similar in quality to those for vertebrate [alpha]-actin and had a critical concentration for assembly of 0.6 [mu]M. Moreover, pollen actin interacted specifically and in a characteristic fashion with several ABPs. Tradescantia cells were microinjected and used as an experimental system to study the behavior of pollen actin in vivo. We demonstrated that purified pollen actin ameliorated the effects of injecting excess profilin into live stamen hair cells.

Tissue‐specific biomass recalcitrance in corn stover pretreated with liquid hot‐water: Enzymatic hydrolysis (part 1)

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue have a measurable effect on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. The first part of our work combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay‐green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. The extent of enzyme hydrolysis follows the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Physical fractionation of corn stalks or other C4 grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses such as thermal processing, combustion, or recycle to the land from which the corn was harvested. Biotechnol. Bioeng. 2012; 109:390–397.

Involvement of snapdragon benzaldehyde dehydrogenase in benzoic acid biosynthesis.

Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.

Phenotypic plasticity in cell walls of maize brown midrib mutants is limited by lignin composition.

The hydrophobic cell wall polymer lignin is deposited in specialized cells to make them impermeable to water and prevent cell collapse as negative pressure or gravitational force is exerted. The variation in lignin subunit composition that exists among different species, and among different tissues within the same species suggests that lignin subunit composition varies depending on its precise function. In order to gain a better understanding of the relationship between lignin subunit composition and the physico-chemical properties of lignified tissues, detailed analyses were performed of near-isogenic brown midrib2 (bm2), bm4, bm2-bm4, and bm1-bm2-bm4 mutants of maize. This investigation was motivated by the fact that the bm2-bm4 double mutant is substantially shorter, displays drought symptoms even when well watered, and will often not develop reproductive organs, whereas the phenotypes of the individual bm single mutants and double mutant combinations other than bm2-bm4 are only subtly different from the wild-type control. Detailed cell wall compositional analyses revealed midrib-specific reductions in Klason lignin content in the bm2, bm4, and bm2-bm4 mutants relative to the wild-type control, with reductions in both guaiacyl (G)- and syringyl (S)-residues. The cellulose content was not different, but the reduction in lignin content was compensated by an increase in hemicellulosic polysaccharides. Linear discriminant analysis performed on the compositional data indicated that the bm2 and bm4 mutations act independently of each other on common cell wall biosynthetic steps. After quantitative analysis of scanning electron micrographs of midrib sections, the variation in chemical composition of the cell walls was shown to be correlated with the thickness of the sclerenchyma cell walls, but not with xylem vessel surface area. The bm2-bm4 double mutant represents the limit of phenotypic plasticity in cell wall composition, as the bm1-bm2-bm4 and bm2-bm3-bm4 mutants did not develop into mature plants, unlike the triple mutants bm1-bm2-bm3 and bm1-bm3-bm4.

Expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies C11E9 and EM-7G1 under acid-, salt- or temperature-induced stress environments

Aims: To study the expression of cellular antigens of Listeria monocytogenes that react with monoclonal antibodies (MAbs) C11E9 and EM‐7G1 under acid‐, salt‐ or temperature‐induced stress environments.

GROWTH, PHYSIOLOGY, AND ULTRASTRUCTURE OF A DIAZOTROPHIC CYANOBACTERIUM, CYANOTHECE SP. STRAIN ATCC 51142, IN MIXOTROPHIC AND CHEMOHETEROTROPHIC CULTURES1

The growth, physiology, and ultrastructure of the marine, unicellular, diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142, was examined under mixotrophic and chemoheterotrophic conditions. Several organic substrates were tested for the capacity to support heterotrophic growth. Glycerol was the only substrate capable of enhancing mixotrophic growth in the light and supporting chemoheterotrophic growth in the dark. Dextrose enhanced mixotrophic growth but could not support chemoheterotrophic growth. Chemoheterotrophic cultures in continuous darkness grew faster and to higher densities than photoautotrophic cultures, thus demonstrating the great respiratory capacity of this cyanobacterial strain. Only small differences in the pigment content and ultrastructure of the heterotrophic strains were observed in comparison to photoautotrophic control strains. The chemoheterotrophic strain grown in continuous darkness and the mixotrophic strain grown in light/dark cycles exhibited daily metabolic oscillations in N2 fixation and glycogen accumulation similar to those manifested in photoautotrophic cultures grown in light/dark cycles or continuous light. This “temporal separation” helps protect O2‐sensitive N2 fixation from photosynthetic O2 evolution. The rationale for cyclic glycogen accumulation in cultures with an ample source of organic carbon substrate is unclear, but the observation of similar daily rhythmicities in cultures grown in light/dark cycles, continuous light, and continuous dark suggests an underlying circadian mechanism.

Tissue‐specific biomass recalcitrance in corn stover pretreated with liquid hot‐water: SEM imaging (part 2)

In the first part of our work, we combined compositional analysis, pretreatment and enzyme hydrolysis for fractionated pith, rind, and leaf tissues from a hybrid stay‐green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis experiments coupled with chemical analysis of the different fractions of corn stover showed significant differences in cell wall structure before and after liquid hot water pretreatment. The extent of enzyme hydrolysis followed the sequence rind < leaves < pith with 90% conversion of cellulose to glucose in 24 h in the best cases. Since similar lignin contents remained after liquid hot water pretreatment of leaves, rind, and pith, our results indicated that the amount of lignin alone is not sufficient to explain the different enzymatic hydrolysis characteristics of the fractions. While the role of structural characteristics on enzyme hydrolysis of cell walls is measured as described in part I, the SEM images presented in this part II of our work show that sugar yields from enzymatic hydrolysis of corn fractions correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment. Biotechnol. Bioeng. 2012; 109:398–404.

HETEROCYST DEVELOPMENT AND LOCALIZATION OF CYANOPHYCIN IN N2-FIXING CULTURES OF ANABAENA SP. PCC 7120 (CYANOBACTERIA)

The process of N2 fixation in the filamentous cyanobacterium Anabaena sp. PCC 7120 is known to occur in terminally differentiated cells called heterocysts. This study is concerned with a morphological and immunocytochemical analysis of the developing heterocysts. The heterocysts continue a developmental process after synthesis of the specialized cell wall and the formation of the proheterocyst. The initial stages were described by Wilcox et al. (1973) and designated stages 1 through 7, with stages 5–7 associated with the maturing heterocyst. We now designate a stage 8 as the postmaturation stage, based on physiological and ultrastructural evidence. Immunocytochemistry to detect the nitrogenase protein NifH and the nonribosomally synthesized polypeptide cyanophycin demonstrated a complementary accumulation of these polypeptides. Accumulation of the nitrogenase protein was greatest at stages 5 and 6 and then declined precipitously. Cyanophycin was more prevalent after late stage 6 and was primarily associated with the polar nodule (polar plug) and the neck connecting the heterocyst with the adjoining vegetative cell. We suggest that the cyanophycin‐containing polar plug is a key intermediate in the storage of fixed nitrogen in the heterocyst, a result consistent with the suggestion first made by Carr (1988) that cyanophycin exists as a dynamic reservoir of fixed nitrogen within the heterocysts.

Metabolic rhythms of a diazotrophic cyanobacterium, cyanothece sp. strain atcc 51142, heterotrophically grown in continuous dark

Aerobic unicellular cyanobacteria temporally separate nitrogen fixation and photosynthetic activity to protect oxygen‐sensitive nitrogenase. Diurnal metabolic oscillations in the unicellular diazotroph, Cyanothece sp. str. CGD occurred even when cells were grown under continuous dark conditions, as determined in this study. The metabolic processes of nitrogen fixation, respiration, carbohydrate storage, and nitrogen storage all remained highly synchronized during heterotrophic growth, despite the absence of light signals. This shows the importance of circadian regulation in this unicellular cyanobacterium. The nitrogenase complex is apparently tightly regulated at the transcriptional and post‐translational levels. Carbohydrates accumulated during the subjective light phase were metabolized in a burst of respiration in the subjective dark phase. The utilization of carbohydrates occurred at the same time as the intense period of nitrogenase activity and downregulation of the capacity for photosynthetic oxygen evolution. Abundance of the D1 protein of photosystem II in Cyanothece sp. str. CGD remained relatively constant under continuous darkness, but the psbA transcript was more abundant in the subjective light phase. Accumulation of the psaAB gene products in photosystem I was highest in the subjective dark phase. The storage of fixed nitrogen as cyanophycin granules also exhibited periodic changes, with the amount of cyanophycin being greatest after the peak of nitrogenase activity. These results strongly implicate a circadian regulatory mechanism operating on these metabolic processes, independent of light cues, and stress the importance of circadian rhythms in global metabolic regulation in this unicellular cyanobacterium.

Composition of the carbohydrate granules of the cyanobacterium, Cyanothece sp. strain ATCC 51142

Cyanothece sp. strain ATCC 51142 is an aerobic, unicellular, diazotrophic cyanobacterium that temporally separates O2-sensitive N2 fixation from oxygenic photosynthesis. The energy and reducing power needed for N2 fixation appears to be generated by an active respiratory apparatus that utilizes the contents of large interthylakoidal carbohydrate granules. We report here on the carbohydrate and protein composition of the granules of Cyanothece sp. strain ATCC 51142. The carbohydrate component is a glucose homopolymer with branches every nine residues and is chemically identical to glycogen. Granule-associated protein fractions showed temporal changes in the number of proteins and their abundance during the metabolic oscillations observed under diazotrophic conditions. There also were temporal changes in the protein pattern of the granule-depleted supernatant fractions from diazotrophic cultures. None of the granule-associated proteins crossreacted with antisera directed against several glycogen-metabolizing enzymes or nitrogenase, although these proteins were tentatively identified in supernatant fractions. It is suggested that the granule-associated proteins are structural proteins required to maintain a complex granule architecture.