Search

The plant kingdom supports an extraordinary chemical diversity, with terpenoids representing a particularly diversified class of secondary (or specialized) metabolites. Volatile and semi-volatile terpenoids in the C10-C20 range are often formed in specialized cell types and secretory structures. In the angiosperm lineage, glandular trichomes play an important role in enabling the biosynthesis and storage (or in some cases secretion) of functionalized terpenoids. The 'decoration' of a terpenoid scaffold with functional groups changes its physical and chemical properties, and can therefore affect the perception of a specific metabolite by other organisms. Because of the ecological implications (e.g. plant-herbivore interactions) and commercial relevance (e.g. volatiles used in the flavor and fragrance industries), terpenoid functionalization has been researched extensively. Recent successes in the cloning and functional evaluation of genes as well as the structural and biochemical characterization of enzyme catalysts have laid the foundation for an improved understanding of how pathways toward functionalized monoterpenes may have evolved. In this review, we will focus on an up-to-date account of functionalization reactions present in glandular trichomes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The pectin matrix of the angiosperm cell wall is regulated in both synthesis and modification and greatly influences the direction and extent of cell growth. Pathogens, herbivory and mechanical stresses all influence this pectin matrix and consequently plant form and function. The cell wall-associated kinases (WAKs) bind to pectin and regulate cell expansion or stress responses depending upon the state of the pectin. This review explores the WAKs in the context of cell wall biology and signal transduction pathways., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.

Pyruvate,orthophosphate dikinase (PPDK) plays a controlling role in the PEP-regeneration phase of the C(4) photosynthetic pathway. Earlier studies have fully documented its biochemical properties and its post-translational regulation by the PPDK regulatory protein (PDRP). However, the question of its evolution into the C(4) pathway has, until recently, received little attention. One assumption concerning this evolution is that changes in catalytic and regulatory properties of PPDK were necessary for the enzyme to fulfil its role in the C(4) pathway. In this study, the functional evolution of PPDK from its ancient origins in the Archaea to its ascension as a photosynthetic enzyme in modern C(4) angiosperms is reviewed. This analysis is accompanied by a comparative investigation into key catalytic and regulatory properties of a C(3) PPDK isoform from Arabidopsis and the C(4) PPDK isoform from Zea mays. From these analyses, it is proposed that PPDK first became functionally seated in C(3) plants as an ancillary glycolytic enzyme and that its transition into a C(4) pathway enzyme involved only minor changes in enzyme properties per se.

In oil palm (Elaeis guineensis Jacq.), approximately 5% of somatic embryo-derived regenerants show homeotic changes during floral development, involving an apparent feminization of male parts in flowers of both sexes, called the 'mantled' phenotype. This variant phenotype is associated with a reduction in the level of global DNA methylation. To explore possible relationships between DNA methylation level and accumulation of DNA-(cytosine-5) methyltransferase (DNMT) transcripts, the full-length coding sequences corresponding to three different DNMT families in oil palm, namely the MET, CMT, and DRM classes, have been isolated and characterized. The corresponding genes were designated as EgMET1, EgCMT1, and EgDRM1, and encode predicted polypeptides of 1543, 925, and 591 amino acid residues, respectively. Expression of oil palm DNMTs was compared between normal and variant calli and inflorescence tissues using quantitative reverse-transcription PCR. A consistent increase in transcript levels of EgMET1 and EgCMT1 was found in variant fast-growing calli relative to nodular-compact calli. Nodular-compact calli give rise to about 5% of abnormal regenerants whereas fast-growing calli generate 95% of 'mantled' palms in their clonal offspring and were previously demonstrated as having markedly hypomethylated DNA. In immature abnormal inflorescences only EgMET1 transcript levels were increased, while no changes in relative abundance of the EgCMT1 or EgDRM1 transcripts were observed. Therefore, the genome-wide hypomethylation previously described in 'mantled' material cannot be explained by a decrease in expression levels of the de novo or maintenance DNMTs, a paradox which has been previously reported in tumour cells, where there is evidence for global hypomethylation of DNA.

The annotation of novel plant genes is frequently based on sequence and structural similarity to known protein motifs. Understanding the biological function of these genes is dependent on identifying conditions under which they are activated, however. The resurrection plant, Xerophyta humilis is a good model system for identifying and characterizing genes which are important for desiccation tolerance. Desiccation induced-1 (dsi-1(VOC)), a previously uncharacterized plant gene, is up-regulated during desiccation in leaves, roots, and seeds in X. humilis. The X. humilis desiccation induced-1 gene, Xhdsi-1(VOC), shares structural homology with the vicinal oxygen chelate (VOC) metalloenzyme superfamily. Proteins in this superfamily share little sequence similarity, but are characterized by a common betaalphabetabetabeta structural fold. A number of plant orthologues of XhDsi-1(VOC) have been identified, including Arabidopsis thaliana At1g07645, which is currently annotated as a glyoxalase I-like gene, and many ESTs derived from seed cDNA libraries. Xhdsi-1(VOC) and its orthologues do not, however, contain the glutathione and zinc binding sites conserved in glyoxalase I genes. Furthermore, expression of Xhdsi-1(VOC) in yeast failed to rescue a yeast glyoxalase I mutant. Messenger RNA transcripts for At1g07645 accumulate during seed maturation, but are not induced by water loss, salt or mannitol stress in vegetative tissue in Arabidopsis. It is concluded that dsi-1(VOC) is a seed-specific gene in desiccation-sensitive plants that is activated by water loss in vegetative tissues in the resurrection plant X. humilis and plays an important role in allowing plant tissues to survive loss of 95% of their relative water content.

The intensity of flower colour, mainly determined by the amount of anthocyanin, is an important horticultural trait. To modulate flower colour intensity, post-transcriptional gene silencing (PTGS)-based technology has been widely used. The constraint of PTGS, however, is that it requires a high degree of conservation in the nucleotide sequences of the target and the silencer. Further, it is difficult to restrict PTGS to the desired tissue or organ due to its systemic spread. To overcome these problems, dominant-negative chalcone synthase (CHS) enzymes have been developed by mutating a cysteine that is essential for the catalytic activity and a methionine that protrudes into the adjoining CHS monomer, as shown through crystallography. The dominant-negative action of mutated CHS enzymes from Mazus japonicus are demonstrated using transgenic Arabidopsis. Also, the modulation of Petunia flower colour intensity by the dominant-negative CHS is shown. The data support the crystallography result showing the importance of the protruding methionine for the function of the adjoining CHS monomer. Furthermore, the modulation of anthocyanin production by the mutated Mazus CHS in Arabidopsis and petunia suggests that the dominant-negative CHS can be used even in distantly related species.

The genus Nepenthes represents carnivorous plants with pitcher traps capable of efficient prey capture and digestion. The possible involvement of plant chitinases in this process was studied in Nepenthes khasiana. Two different types of endochitinases were identified in the liquid of closed traps exhibiting substrate specificity for either long chitin polymers or N-acetylglucosamine (GlcNAc) oligomers. Injection of chitin into such closed sterile pitchers induced the appearance of additional endochitinase isoenzymes, with substrate specificity only for long chitin polymers. No significant exochitinase (N-acetyl-beta-glucosaminidase) or chitobiosidase activity could be detected in the non-induced or induced trap liquid. Four genes representing two subgroups of basic chitinases, denoted as Nkchit1b and Nkchit2b, were isolated from the secretory region of N. khasiana pitchers. The main differences between the two subgroups are the presence of a proline-rich hinge region only in NkCHIT1b and a C-terminal putative vacuole targeting extension only in NkCHIT2b, indicating different compartmentalization of the two enzymes. Reverse transcription-polymerase chain reaction (RT-PCR) evaluation of mRNA levels showed that the Nkchit2b genes are constitutively expressed in the secretory cells while transcription of Nkchit1b genes is induced by chitin injection. These results show for the first time the involvement of genes encoding chitinases in prey-trap interaction and their differential expression and activity during prey trapping.

The developmental pathway of somatic embryogenesis in Norway spruce involves proliferation of proembryogenic masses (PEMs), PEM-to-somatic embryo transition and further development of the somatic embryos. It has previously been shown that extracellular signal molecules, including arabinogalactan proteins, lipo-chitooligosaccharides and chitinases, regulate somatic embryogenesis. The Chia4-Pa1 gene from Norway spruce is described here. The Chia4-Pa1 encodes a typical basic class IV chitinase, although the intron-exon organization of this gymnosperm chitinase is different from that in angiosperm class IV chitinases. The Chia4-Pa1 belongs to a small gene family with highly similar members, and the expression pattern of Chia4-Pa1 cannot be distinguished from that of other Chia4-Pa members. Upon withdrawal of plant growth regulators, i.e. during a treatment that stimulates PEM-to-somatic embryo transition and massive programmed cell death, a significant increase in transcription and translation of Chia4-Pa genes takes place. The expression pattern analysis revealed that Chia4-Pa genes are expressed in a subpopulation of proliferating cells and at the base of the somatic embryo. Furthermore, in seeds, Chia4-Pa genes are expressed in the megagametophyte in the single cell-layered zone surrounding the corrosion cavity. Taken together these results suggest that the Chia4-Pa expressing cells have a megagametophyte signalling function and that CHIA4-Pa stimulates programmed cell death and promotes PEM-to-somatic embryo transition.

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.

Ammonium is assimilated into amino acids through the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) enzymes. This metabolic pathway is driven by energy, reducing power and requires the net supply of 2-oxoglutarate that can be provided by the reaction catalysed by isocitrate dehydrogenase (IDH). Most studies on the biochemistry and molecular biology of N-assimilating enzymes have been carried out on annual plant species and the available information on woody models is far more limited. This is in spite of their economic and ecological importance and the fact that nitrogen is a common limiting factor for tree growth. GS, GOGAT and IDH enzymes have been purified from several woody species and their kinetic and molecular properties determined. A number of cDNA clones have also been isolated and characterized. Although the enzymes are remarkably well conserved along the evolutionary scale, major differences have been found in their compartmentation within the cell between angiosperms and conifers, suggesting possible adaptations to specific functional roles. The analysis of the gene expression patterns in a variety of biological situations such as changes in N nutrition, development, biotic or abiotic stresses and senescence, suggest that cytosolic GS plays a central and pivotal role in ammonium assimilation and metabolism in woody plants. The modification of N assimilation efficiency has been recently approached in trees by overexpression of a cytosolic pine GS in poplar. The results obtained, suggest that an increase in cytosolic GS might lead to a global effect on the synthesis of nitrogenous compounds in the leaves, with enhanced vegetative growth of transgenic trees. All these data suggest that manipulation of cytosolic GS may have consequences for plant growth and biomass production.

Though endopeptidases and carboxypeptidases are present in protein bodies of dry quiescent seeds the function of these proteases during germination is still a matter of debate. In some plants it was demonstrated that endopeptidases of dry protein bodies degrade storage proteins of these organelles. Other studies describe cases where this did not happen. The role that stored proteinases play in the initiation of storage protein breakdown in germinating seeds thus remains unclear. Numerous reviews state that the initiation of reserve protein mobilization is attributed to de novo formed endopeptidases which together with stored carboxypeptidases degrade the bulk of proteins in storage organs and tissues after seeds have germinated. The evidence that the small amounts of endopeptidases in protein bodies of embryonic axes and cotyledons of dry seeds from dicotyledonous plants play an important role in the initiation of storage protein mobilization during early germination is summarized here.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.

A wild-type poplar hybrid and two transgenic clones overexpressing a bacterial gamma-glutamylcysteine synthetase in the cytosol or in the chloroplasts were exposed to the chloroacetanilide herbicides acetochlor and metolachlor dispersed in the soil. The transformed poplars contained higher gamma-glutamylcysteine and glutathione (GSH) levels than wild-type plants and therefore it was supposed that they would have an elevated tolerance towards these herbicides, which are detoxified in GSH-dependent reactions. Phenotypically, the transgenic and wild-type plants did not differ. The growth and the biomass of all poplar lines were markedly reduced by the two chloroacetanilide herbicides. However, the decrease of shoot and root fresh weights caused by the herbicides was significantly smaller in the transgenic than in wild-type plants. In addition, the growth rate of poplars transformed in the cytosol was reduced to a significantly lesser extent than that of wild-type plants following herbicide treatments. The effects of the two herbicides were similar. Herbicide exposures markedly increased the levels of gamma-glutamylcysteine and GSH in leaves of each poplar line. The increase in the foliar amounts of these thiols was stronger in the transgenic lines than in the wild type, particularly in the upper leaves. Considerable GST activities were detected in leaves of all poplar plants. Exposure of poplars to chloroacetanilide herbicides resulted in a marked induction of GST activity in upper leaf positions but not in middle and lower leaves. The extent of enzyme induction did not differ significantly between transgenic and wild-type poplars. Although the results show that the transgenic poplar lines are good candidates for phytoremediation purposes, the further improvement of their detoxification capacity, preferably by transformation using genes encoding herbicide-specific GST isoenzymes, seems to be the most promising way to obtain plants suitable for practical application.

In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells.

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immunopurification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H(+)-ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacylglycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.

Heterozygous mutants of Amaranthus edulis deficient in PEP carboxylase (PEPC) have been used to study the control of photosynthetic carbon assimilation. A reduction in PEPC activity led to a decrease in the initial slope of the relationship between the CO2 assimilation rate and the intercellular CO2 concentration and to a decrease in photosynthesis at high light intensities, consistent with a decrease in the capacity of the C4 cycle in high light. PEPC exerted appreciable control on photosynthetic flux in the wild-type, with a relatively high flux control coefficient of 0.35 in saturating light and ambient CO2. The flux control coefficient was decreased in low light or increased in low CO2 or in plants containing lower PEPC activity. However, the rate of CO2 assimilation decreased down to about 55% PEPC, followed by an up-turn in the light-saturated photosynthetic rate as PEPC was further reduced, suggesting the existence of a mechanism that compensates for the loss of PEPC activity. The amounts of photosynthetic metabolites, including glycine and serine, also showed a biphasic response to decreasing PEPC. There was a linear relationship between the activity of PEPC and the activation state of the enzyme. A possible mechanism of compensation involving photorespiratory intermediates is discussed.