Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.Lignin is a major structural and protective component of plant cell walls. Lignin exists as a polymer of mainly three hydroxycinnamyl alcohols and related compounds, referred to as monolignols. The most common monolignols are coniferyl, sinapyl, and p-coumaryl alcohol (Ralph et al., 2004; Vanholme et al., 2010). After polymerization, structures derived from those compounds are referred to as guaiacyl, syringyl, and p-hydroxyphenyl subunits, respectively. The specific composition of lignin subunits varies among species, tissues, and developmental stages. Gymnosperm trees produce lignin that is primarily made of guaiacyl subunits, angiosperm trees contain guaiacyl and syringyl subunits, whereas grasses contain guaiacyl and syringyl subunits with small amounts (approximately 5%) of p-hydroxyphenyl residues. This observed variation in subunit composition across species may reflect the heterogeneity in substrate specificity and kinetic parameters among various monolignol biosynthetic enzymes (Weng et al., 2008).Biosynthesis of the monolignols occurs via the phenylpropanoid pathway using Phe precursors (Vanholme et al., 2010). Phe ammonia lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from Phe (Vanholme et al., 2010). Grasses can bypass cinnamate-4-hydroxylase by using Tyr as a substrate for Phe ammonia lyase (Neish, 1961; Rösler et al., 1997). The hydroxycinnamoyltransferase (HCT) enzymes exchange the CoA functionality esterified to p-coumaric acid with shikimic or quinic acid to allow for the subsequent conversion of the p-coumaroyl moiety to a caffeoyl moiety by p-coumarate-3′-hydroxylase (C3′H). The hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferases (HSTs) exhibit preference for shikimate, whereas the hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferases prefer quinate as a substrate (Sander and Petersen, 2011). Subsequent reactions ultimately lead to coniferyl and sinapyl alcohol via reduction of the γ-carbon on the propane side chain and substitution of the C3 and C5 positions of the phenol ring (Boerjan et al., 2003).Sorghum (Sorghum bicolor) is an attractive bioenergy crop with typical dry biomass yields between 20 and 25 Mg ha⁻¹ and yields as high as 40 Mg ha⁻¹ possible under optimal conditions (Venuto and Kindiger, 2008). Moreover, sorghum utilizes nitrogen-based fertilizer more efficiently than maize (Zea mays) and sugarcane (Saccharum officinarum), leading to less groundwater contamination and lower CO₂ emission (Propheter and Staggenborg, 2010; Wortmann and Regassa, 2011). Overall, sorghum has a higher sugar yield potential per land area and requires less water for growth than maize, allowing it to grow in a more diverse range of environments (Saballos, 2008). The sorghum genome sequence has been released (Paterson et al., 2009), and Targeting Induced Local Lesions in Genomes populations exist (Xin et al., 2008) in which various cell wall mutants have been identified (Sattler et al., 2012; Vermerris and Saballos, 2012).A detailed understanding of the catalytic mechanism of phenylpropanoid-related enzymes will enable the targeted modification of lignin subunit composition. The presence of lignin poses a major obstacle to the production of biofuels and chemicals from lignocellulosic biomass, because of its ability to hinder the activity of enzymes required to degrade cellulose to sugars that can be fermented for ethanol production (Yang and Wyman, 2004; Berlin et al., 2006). Genetic modification of plant cell wall composition, especially lignin content and subunit composition, has been shown to improve biomass conversion to fermentable sugars (Chen and Dixon, 2007; Vermerris et al., 2007; Jung et al., 2012). In particular, HCT silencing in Arabidopsis (Arabidopsis thaliana) causes an accumulation of p-hydroxyphenyl residues in the lignin and decreased content of guaiacyl and syringyl residues, leading to a dwarf phenotype (Li et al., 2010). Down-regulation of HCT has also been shown to result in decreased plant growth in alfalfa (Medicago sativa; Shadle et al., 2007). Concomitantly, ruminant digestibility and the yield of fermentable sugars following enzymatic saccharification increased (Chen and Dixon, 2007; Shadle et al., 2007). Reduced HCT activity may alter cell wall polymer interactions and allow better access of cellulolytic enzymes to the cellulose. Therefore, it has the potential to reduce the energy and processing costs associated with the conversion of biomass to fuels and chemicals. However fine-tuning will be necessary to limit the negative impacts on plant growth, which will require a detailed understanding of the catalytic mechanism of HCT.Given the difference in lignin subunit composition among different species and the prominence of grasses among dedicated bioenergy crops, we have focused on elucidating the crystal structure and activity of monolignol-related enzymes of sorghum, starting with the HST-like HCT. HCT belongs to the BAHD superfamily of plant-specific acyl-CoA-dependent acyltransferases (Ma et al., 2005; D’Auria, 2006). However, the BAHD superfamily has functionally and structurally diverse members that frequently possess little (as low as 10%) sequence identity among them (St-Pierre and Luca, 2000). Recent studies led to the crystal structure of the HST-like HCT from robusta coffee (Coffea canephora), an angiosperm dicot with a binding pocket elucidated by molecular docking and mutagenesis (Lallemand et al., 2012). In this report, we present the three-dimensional structures of HCT in its apo-form and ternary complex, supplemented by mutagenic studies to elucidate its reaction mechanism and structural relationship to other members in this growing functional class.     

Respiratory C fluxes and root exudation differ in two full-sib clones of Pinus taeda (L.) under contrasting fertilizer regimes in a greenhouse

Aims

We investigated whether changes in respiratory C fluxes, soil CO₂ efflux, or root exudate quantity or quality explained differences in growth rates between closely related clones of Pinus taeda (L.).

Methods

A factorial design with two clones, fertilized and control treatments, and four sequential harvests was installed in a greenhouse for 121 days.

Results

The two clones did show significant differences in respiratory C fluxes, soil CO₂ efflux, and root exudation quantity and quality. While the clones also differed in growth rates, the C fluxes assessed in this paper did not explain how seedlings were able to allocate more C to stem growth in the months following fertilizer application. Changes in root exudation were not consistent with reduced heterotrophic soil CO₂ efflux, which does not appear to be a plant-mediated process.

Conclusions

These results indicate that if single genotypes are deployed over large land areas in plantations, dramatic differences between clonal plant-soil interactions may require consideration in ecosystem C budgets. Further, the range of belowground fluxes observed implies that genotype-specific C allocation may make some clones better able to exploit a given resource environment than others.     

DR3/LARD spliced mRNA variants’ frequency in colorectal cancer

Many variants of the DR3/LARD death receptor mRNA are derived during alternative splicing. Different DR3/LARD mRNAs encode the membrane and soluble forms of the receptor, which perform different functions. The frequency of the spliced mRNA variants of DR3/LARD was assessed by RT-PCR in patients with colorectal cancer and in cancer cell lines. Four forms of the DR3/LARD death receptor mRNA were detected with different frequencies in the studied samples. Two of them encoded the membrane molecules (LARD 1a mRNA and DR3β mRNA) and two other forms expressed the soluble forms of the receptor (LARD 3 mRNA and soluble DR3β mRNA). In the blood of healthy volunteers, 11 variants (spectra) of DR3/LARD mRNA forms were identified, and the full spectrum that included all four variants of DR3/LARD mRNA dominated. In blood and tumor center samples from patients with colon cancer, six spectra of DR3/LARD mRNA were found. The diversity of the DR3/LARD mRNA spectra was decreased in colon cancer patients due to the reduced frequency of soluble DR3β mRNA. In samples of tumor centers, the spectrum with the absence of only mRNA of the soluble DR3β form dominated. In the blood of patients, two spectra prevailed, i.e., the full spectrum and LARD 1a mRNA and LARD 3 mRNA. Only these two spectra of DR3/LARD mRNA were also found in cancer cell lines. Distinctions in the frequency of DR3/LARD mRNA spectra in healthy volunteers and patients with colorectal cancer can define the different susceptibility of immunocompetent and tumor cells to apoptosis signals.     

DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.     

The carnivorous plant described as Sarracenia alata contains two cryptic species

Modern methods for species delimitation provide biologists with the power to detect cryptic diversity in nearly any system. To illustrate the application of such methods, we collected data (21 sequence loci) from a carnivorous plant in southeastern North America and applied several recently developed methods (Gaussian clustering, Structurama, BPP, spedeSTEM). The pale pitcher plant Sarracenia alata inhabits the southeastern USA along the northern coast of the Gulf of Mexico. Sarracenia alata populations are separated by the Mississippi River and Atchafalaya Basin, a known biogeographical barrier in this region, but the cohesiveness of S. alata as currently classified has not been tested rigorously. Multiple analytical approaches (including allelic clustering and species trees methods) suggest that S. alata comprises two cryptic lineages that correspond to the eastern and western portions of the plant's distribution. That such clear genetic evidence for cryptic diversity exists within S. alata and is in conflict with other sources of data (e.g. morphology, environmental differentiation) illustrates a conundrum faced by those who investigate species boundaries: genetic data are often the first type of data to accumulate evidence of differentiation, but most existing taxonomic treatments are based on nongenetic data. Our results suggest that S. alata as currently described contains two cryptic species, and we recommend the elevation of the western populations to species status. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 737–746.     

Geometric morphometrics as a tool for interpreting evolutionary transitions in the black fly wing (Diptera: Simuliidae)

A geometric morphometric analysis was conducted on wing‐vein landmarks on exemplar species of the family Simuliidae of the following genera: Parasimulium, Gymnopais, Twinnia, Helodon, Prosimulium, Greniera, Stegopterna, Tlalocomyia, Cnephia, Ectemnia, Metacnephia, Austrosimulium, and Simulium. Generalized least squares superimposition was performed on landmarks, followed by a principal component analysis on resulting Procrustes distances. Patterns of shape change along the principal component axes were visualized using the thin‐plate spline. The analysis revealed wing shape diversity through (1) the insertion points of the subcosta and R₁, resulting in the terminus of the costa exhibiting a trend towards a more apical position on the wing, and (2) the insertion point of the humeral cross vein, resulting in the anterior branch of the media exhibiting a trend toward a more basal position on the wing. Canonical variates analysis of Procrustes distances successfully assigned all exemplar species into their a priori taxonomic groupings. The diversity in wing shape reveals a trend towards decreased length of basal radial cell and increased costalization of anterior wing veins in the evolutionary transition from plesiomorphic prosimuliines to more derived simuliines. The functional significance of these evolutionary transitions is discussed. © 2013 The Linnean Society of London     

OBSERVATIONS ON THE SLATY EGRET EGRETTA VINACEIGULA IN NORTHERN NAMIBIA

Hines, C.J.H. 1992. Observations on the Slaty Egret Egretta vinaceigula in northern Namibia. Ostrich 63: 118–122.

The Slaty Egret is an endemic African resident with a limited distribution in Botswana, Zambia and Namibia. Distribution records for Namibia are reviewed and information on movements and habitat preferences is given. Diet and feeding habits are described. Two colonies of Slaty Egrets containing eggs and chicks were found on temporary wetlands in eastern Bushmanland. Nests and eggs are described and egg measurements and weights given. The plumage of immature birds is described as well as the variation in adult plumages. The conservation status of the species is reviewed, and the possible importance of temporary wetlands to the Slaty Egret is discussed.     

AVIAN NOMENCLATURAL ISSUES ARISING FROM THE PUBLICATION OF ROOKMAAKER'S THE ZOOLOGICAL EXPLORATION OF SOUTHERN AFRICA 1650–1790

Clancey, P. A. & Brooke, R. K. 1990. Avian nomenclatural issues arising from the publication of Rook-maaker's The Zoological Exploration of Southern Africa 1650–1790. Ostrich 61:143-145.

Rookmaaker (1989) has drawn the attention of taxonomists to the existence of a range of long overlooked senior ornithological synonyms which, if adopted in terms of the Law of Priority, would result in changes to long established names. As such extensive changes of name are patently undesirable, we propose a course of action which would entail as little disruption as possible to current usage, and obviate the necessity for the submission of cases to the International Commission on Zoological Nomenclature.     

6. ANOTHER NOTE ON SAROTHRURA ELEGANS

Summary

Dowsett-Lemaire, F. 1983. Studies of a breeding population of Waller's Redwinged Starlings in montane forests of south-central Africa. Ostrich. 54:105-112.

The breeding biology and behaviour of Waller's Redwinged Starling Onychognuthus walleri was studied for two seasons in forest patches on the Nyika Plateau, 2 200 m ad. (Mala?i-Zambia). In general behaviour (e.g. strong pair bond, fidelity to the nest site), O. walleri resembles other species of the genus. Twenty-six egg-laying records are from late August to mid-November (i.e. end of dry season to early rains). Suitable nest sites (natural holes in trees) seem to be in short supply, and the non-breeding population is far in excess of the breeding one. Incubation periods lasted 13 days (2) to 16 days (1). One nestling period was 29 days. Insects are more important than fruits in the diet of nestlings. Annual breeding productivity is low (0,5-0,6 young/breeding pair) because of heavy nest predation, and at tunes insect-food shortages leading to starvation of young. There were few re-laying attempts, and only one abnormal attempt at a second brood.

Two other congeneric species, the Slenderbilled Redwinged Starling O. tenuirostris and the African Redwinged Starling O. morio also breed on the Nyika. Notes on their feeding habits, numbers and nest sites are given. One nest site of O. tenuirostris on a wet rock face by waterfalls was taken over by O. morio in the following season.