Thomas H. Roberts

Techniques for Analysis of Plant Phenolic Compounds

Phenolic compounds are well-known phytochemicals found in all plants. They consist of simple phenols, benzoic and cinnamic acid, coumarins, tannins, lignins, lignans and flavonoids. Substantial developments in research focused on the extraction, identification and quantification of phenolic compounds as medicinal and/or dietary molecules have occurred over the last 25 years. Organic solvent extraction is the main method used to extract phenolics. Chemical procedures are used to detect the presence of total phenolics, while spectrophotometric and chromatographic techniques are utilized to identify and quantify individual phenolic compounds. This review addresses the application of different methodologies utilized in the analysis of phenolic compounds in plant-based products, including recent technical developments in the quantification of phenolics.

Serpins in plants and green algae.

Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.

Inhibitory Serpins from Wheat Grain with Reactive Centers Resembling Glutamine-rich Repeats of Prolamin Storage Proteins CLONING AND CHARACTERIZATION OF FIVE MAJOR MOLECULAR FORMS

Genes encoding proteins of the serpin superfamily are widespread in the plant kingdom, but the properties of very few plant serpins have been studied, and physiological functions have not been elucidated. Six distinct serpins have been identified in grains of hexaploid bread wheat (Triticum aestivum L.) by partial purification and amino acid sequencing. The reactive centers of all but one of the serpins resemble the glutamine-rich repetitive sequences in prolamin storage proteins of wheat grain. Five of the serpins, classified into two protein Z subfamilies, WSZ1 and WSZ2, have been cloned, expressed in Escherichia coli, and purified. Inhibitory specificity toward 17 proteinases of mammalian, plant, and microbial origin was studied. All five serpins were suicide substrate inhibitors of chymotrypsin and cathepsin G. WSZ1a and WSZ1b inhibited at the unusual reactive center P1-P1′ Gln-Gln, and WSZ2b at P2-P1 Leu-Arg—one of two overlapping reactive centers. WSZ1c with P1-P1′ Leu-Gln was the fastest inhibitor of chymotrypsin (k a = 1.3 × 106 m −1 s−1). WSZ1a was as efficient an inhibitor of chymotrypsin as WSZ2a (k a∼105 m −1 s−1), which has P1-P1′ Leu-Ser—a reactive center common in animal serpins. WSZ2b inhibited plasmin at P1-P1′ Arg-Gln (k a∼103 m −1 s−1). None of the five serpins inhibited Bacillus subtilisin A,Fusarium trypsin, or two subtilisin-like plant serine proteinases, hordolisin from barley green malt and cucumisin D from honeydew melon. Possible functions involving interactions with endogenous or exogenous proteinases adapted to prolamin degradation are discussed.

Strategic Distribution of Protective Proteins within Bran Layers of Wheat Protects the Nutrient-Rich Endosperm

Bran from bread wheat (Triticum aestivum ‘Babbler’) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilizing organic substrates in the endosperm during germination. We microdissected three defined bran fractions, outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa, and nucellar tissue), and inner layer (aleurone cells), and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes, whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in metabolism, as would be expected from live aleurone cells, but this layer also includes defense enzymes and inhibitors as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein I to the xylan-rich nucellar layer of the intermediate fraction and pathogenesis-related protein 4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase, and polyphenol oxidase were highest in the outer layers, whereas chitinase activity was found only in assays of whole grains. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.

Evidence for the presence of two rotenone-insensitive NAD(P)H dehydrogenases on the inner surface of the inner membrane of potato tuber mitochondria

Abstract Submitochondrial particles were isolated from potato (Solanum tuberosum L.) tubers. The latency of cytochrome-c oxidase and succinate dehydrogenase indicated that they were 90% inside-out. The inability of the submitochondrial particles to form a membrane potential inside negative as monitored by safranine absorbance changes and their ability to form a large membrane potential inside positive as monitored by oxonol VI absorbance changes confirmed this sidedness. Through the use of rotenone to inhibit Complex I, and diphenyleneiodonium to inhibit both Complex I (by binding to the FMN in the active site) as well as rotenone-insensitive NADPH oxidation, it was possible to distinguish three different NAD(P)H dehydrogenases on the inner surface of the inner mitochondrial membrane: (1) a rotenone-sensitive, diphenyleneiodonium-sensitive, Ca2+-independent enzyme which prefers NADH as the substrate, i.e., Complex I; (2) a rotenone-insensitive, diphenyleneidoonium-sensitive, Ca2+-dependent NAD(P)H dehydrogenase; (3) a rotenone-insensitive, diphenyleneiodonium-insensitive, Ca2+-independent NADH dehydrogenase. All three enzymes were linked to the electron transport chain before Complex III as shown by antimycin A sensitivity and to proton pumping as shown by the generation of a membrane potential. The possible significance of these three enzymes for the function of the mitochondrion in the plant cell is discussed.

Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria

Exogenous NADPH oxidation by purified mitochondria from both potato tuber and Arum maculatum spadix was completely and irreversibly inhibited by sub‐micromolar diphenyleneiodonium (DPI), while exogenous NADH oxidation was inhibited to only a small degree. Addition of DPI caused the collapse of the membrane potential generated by NADPH oxidation, while the potential generated by NADH was unaffected. We conclude that there are two distint enzymes on the outer surface of the inner membrane of plant mitochondria, one specific for NADH, the other relatively specific for NADPH, with both enzymes linked to the electron transport chain.

Three semidominant barley mutants with single amino acid substitutions in the smallest magnesium chelatase subunit form defective AAA+ hexamers

Many enzymes of the bacteriochlorophyll and chlorophyll biosynthesis pathways have been conserved throughout evolution, but the molecular mechanisms of the key steps remain unclear. The magnesium chelatase reaction is one of these steps, and it requires the proteins BchI, BchD, and BchH to catalyze the insertion of Mg2+ into protoporphyrin IX upon ATP hydrolysis. Structural analyses have shown that BchI forms hexamers and belongs to the ATPases associated with various cellular activities (AAA+) family of proteins. AAA+ proteins are Mg2+-dependent ATPases that normally form oligomeric ring structures in the presence of ATP. By using ATPase-deficient BchI subunits, we demonstrate that binding of ATP is sufficient to form BchI oligomers. Further, ATPase-deficient BchI proteins can form mixed oligomers with WT BchI. The formation of BchI oligomers is not sufficient for magnesium chelatase activity when combined with BchD and BchH. Combining WT BchI with ATPase-deficient BchI in an assay disrupts the chelatase reaction, but the presence of deficient BchI does not inhibit ATPase activity of the WT BchI. Thus, the ATPase of every WT segment of the hexamer is autonomous, but all segments of the hexamer must be capable of ATP hydrolysis for magnesium chelatase activity. We suggest that ATP hydrolysis of each BchI within the hexamer causes a conformational change of the hexamer as a whole. However, hexamers containing ATPase-deficient BchI are unable to perform this ATP-dependent conformational change, and the magnesium chelatase reaction is stalled in an early stage.

Arabidopsis AtSerpin1, crystal structure and in vivo interaction with its target protease RESPONSIVE TO DESICCATION-21 (RD21).

In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 Å. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor l-trans-epoxysuccinyl-l-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the l-trans-epoxysuccinyl-l-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.

Serpins in Unicellular Eukarya, Archaea, and Bacteria: Sequence Analysis and Evolution

Most serpins irreversibly inactivate specific serine proteinases of the chymotrypsin family. Inhibitory serpins are unusual proteins in that their native structure is metastable, and rapid conversion to a relaxed state is required to trap target enzymes in a covalent complex. The evolutionary origin of the serpin fold is unresolved, and while serpins in animals are known to be involved in the regulation of a remarkable diversity of metabolic processes, the physiological functions of homologues from other phyla are unknown. Addressing these questions, here we analyze serpin genes identified in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea, where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics of inhibitory serpins, and where multiple serpin genes are found in one genome, variability is displayed in the region of the reactive-center loop important for specificity. All the unicellular eukaryotic serpins have large hydrophobic or positively charged residues at the putative P1 position. In contrast, none of the prokaryotic serpins has a residue of these types at the predicted P1 position, but many have smaller, neutral residues. Serpin evolution is discussed.

Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates

Aims:  Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest.