Transcriptome-Wide Identification and Quantification of Caffeoylquinic Acid Biosynthesis Pathway and Prediction of Its Putative BAHDs Gene Complex in A. spathulifolius.

The phenylpropanoid pathway is a major secondary metabolite pathway that helps plants overcome biotic and abiotic stress and produces various byproducts that promote human health. Its byproduct caffeoylquinic acid is a soluble phenolic compound present in many angiosperms. Hydroxycinnamate-CoA shikimate/quinate transferase is a significant enzyme that plays a role in accumulating CQA biosynthesis. This study analyzed transcriptome-wide identification of the phenylpropanoid to caffeoylquinic acid biosynthesis candidate genes in A. spathulifolius flowers and leaves. Transcriptomic analyses of the flowers and leaves showed a differential expression of the PPP and CQA biosynthesis regulated unigenes. An analysis of PPP-captive unigenes revealed a major duplication in the following genes: PAL, 120 unigenes in leaves and 76 in flowers; C3'H, 169 unigenes in leaves and 140 in flowers; 4CL, 41 unigenes in leaves and 27 in flowers; and C4H, 12 unigenes in leaves and 4 in flowers. The phylogenetic analysis revealed 82 BAHDs superfamily members in leaves and 72 in flowers, among which five unigenes encode for HQT and three for HCT. The three HQT are common to both leaves and flowers, whereas the two HQT were specialized for leaves. The pattern of HQT synthesis was upregulated in flowers, whereas HCT was expressed strongly in the leaves of A. spathulifolius. Overall, 4CL, C4H, and HQT are expressed strongly in flowers and CAA and HCT show more expression in leaves. As a result, the quantification of HQT and HCT indicates that CQA biosynthesis is more abundant in the flowers and synthesis of caffeic acid in the leaves of A. spathulifolius.

Catalytic Plasticity of Germacrene A Oxidase Underlies Sesquiterpene Lactone Diversification.

Adaptive evolution of enzymes benefits from catalytic promiscuity. Sesquiterpene lactones (STLs) have diverged extensively in the Asteraceae, and studies of the enzymes for two representative STLs, costunolide and artemisinin, could provide an insight into the adaptive evolution of enzymes. Costunolide appeared early in Asteraceae evolution and is widespread, whereas artemisinin is a unique STL appearing in a single Asteraceae species, Artemisia annua Therefore, costunolide is a ubiquitous STL, while artemisinin is a specialized one. In costunolide biosynthesis, germacrene A oxidase (GAO) synthesizes germacrene A acid from germacrene A. Similarly, in artemisinin biosynthesis, amorphadiene oxidase (AMO) synthesizes artemisinic acid from amorphadiene. GAO promiscuity is suggested to drive the diversification of STLs. To examine the degree of GAO promiscuity, we expressed six sesquiterpene synthases from cotton (Gossypium arboretum), goldenrod (Solidago canadensis), valerian (Valeriana officinalis), agarwood (Aquilaria crassna), tobacco (Nicotiana tabacum), and orange (Citrus sinensis) in yeast to produce seven distinct sesquiterpene substrates (germacrene D, 5-epi-aristolochene, valencene, δ-cadinene, α- and δ-guaienes, and valerenadiene). GAO or AMO was coexpressed in these yeasts to evaluate the promiscuities of GAO and AMO. Remarkably, all sesquiterpenes tested were oxidized to sesquiterpene acids by GAO, but negligible activities were found from AMO. Hence, GAO apparently has catalytic potential to evolve into different enzymes for synthesizing distinct STLs, while the recently specialized AMO demonstrates rigid substrate specificity. Mutant GAOs implanted with active site residues of AMO showed substantially reduced stability, but their per enzyme activities to produce artemisinic acid increased by 9-fold. Collectively, these results suggest promiscuous GAOs can be developed as novel catalysts for synthesizing unique sesquiterpene derivatives.

New Methyl Threonolactones and Pyroglutamates of Spilanthes acmella (L.) L. and Their Bone Formation Activities.

In our continuing research for bioactive constituents from natural resources, a new methyl threonolactone glucopyranoside (1), a new methyl threonolactone fructofuranoside (2), 2 new pyroglutamates (3 and 4), and 10 known compounds (5-14) were isolated from the whole plant of Spilanthes acmella (L.) L. The structures of these compounds were determined based on various spectroscopic and chemical analyses. All of the isolated compounds were evaluated on bone formation parameters, such as ALP (alkaline phosphatase) and mineralization stimulatory activities of MC3T3-E1 cell lines. The results showed that the new compound, 1,3-butanediol 3-pyroglutamate (4), 2-deoxy-D-ribono-1,4-lactone (6), methyl pyroglutamate (7), ampelopsisionoside (10), icariside B1 (11), and benzyl α-L-arabinopyranosyl-(1→6)-ß-D-glucopyranoside (12) stimulated both ALP and mineralization activities.

Bioengineering triacetic acid lactone production in Yarrowia lipolytica for pogostone synthesis.

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.

A coupled in vitro/in vivo approach for engineering a heterologous type III PKS to enhance polyketide biosynthesis in Saccharomyces cerevisiae.

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts.

Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae.

The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs. Our previous discovery of GAO activity conserved across all major subfamilies of Asteraceae, including the phylogenetically basal lineage of Barnadesioideae, prompted further investigation of the presence of the gateway GAS in Barnadesioideae. Herein we isolated two terpene synthases (BsGAS1/BsGAS2) from the basal Barnadesia spinosa (Barnadesioideae) that displayed robust GAS activity when reconstituted in yeast and characterized in vitro. Despite the apparent lack of STLs in the Barnadesioideae, this work unambiguously confirms the presence of GAS in the basal genera of the Asteraceae. Phylogenetic analysis reveals that the two BsGASs fall into two distinct clades of the Asteraceae's GASs, and BsGAS1 clade is only retained in the evolutionary closer Cichorioideae subfamily, implicating BsGAS2 is likely the ancestral base of most GASs found in the lineages outside the Barnadesioideae. Taken together, these results show the enzymatic capacities of GAS and GAO emerged prior to the subsequent radiation of STL-producing Asteraceae subfamilies.

Yeast and mould dynamics in Caciofiore della Sibilla cheese coagulated with an aqueous extract of Carlina acanthifolia All.

Caciofiore della Sibilla is a speciality ewes' milk cheese traditionally manufactured in a foothill area of the Marche region (Central Italy) with a crude extract of fresh young leaves of Carlina acanthifolia All. subsp. acanthifolia as a coagulating agent. The fungal dynamics and diversity of this speciality cheese were investigated throughout the manufacturing and 20-day ripening process, using a combined PCR-DGGE approach. The fungal biota of a control ewes' milk cheese, manufactured with the same batch of milk coagulated with a commercial animal rennet, was also monitored by PCR-DGGE, in order to investigate the contribution of the peculiar vegetable coagulant to the fungal diversity and dynamics of the cheese. Based on the overall results collected, the raw milk and the dairy environment represented the main sources of fungal contamination, with a marginal or null contribution of thistle rennet to the fungal diversity and dynamics of Caciofiore della Sibilla cheese. Copyright © 2016 John Wiley & Sons, Ltd.

Anthocyanin biosynthesis in gerbera cultivar 'Estelle' and its acyanic sport 'Ivory'.

MAIN CONCLUSION: Identification of distinct allelic versions for dihydroflavonol 4-reductase in gerbera cultivars reveals that gerbera DFR enzymes have strong substrate preference in vivo that is not reflected to the activity in vitro. Flavonoids in the model ornamental plant Gerbera hybrida consist of flavones, flavonols and anthocyanins. Anthocyanins accumulate in the adaxial epidermis of petals and give the different cultivars their characteristic red and violet colour. Both pelargonidin and cyanidin derivatives are found in gerbera, but none of the cultivars contain delphinidin. 'Ivory', a cultivar with white petals, is a sport of the pelargonidin-containing pink cultivar 'Estelle', i.e. it originates from an acyanic branch of 'Estelle'. In this work, four different alleles encoding dihydroflavonol 4-reductase (DFR) were identified in gerbera cultivars. We found that, in contrast to 'Estelle' with the functional allele GDFR1-2, 'Ivory' carries a mutation in this gene that results in an inactive enzyme. Interestingly, 'Ivory' also expresses a second, nonmutated allele (GDFR1-3) in petal epidermi, leading to extractable DFR activity but not to anthocyanin biosynthesis. The second allele encodes a protein identical in amino acid sequence to the DFR of the cyanidin-containing variety 'President'. Pelargonidin-containing cultivars do not react to the flavonoid 3'-hydroxylase inhibitor tetcyclacis, but cyanidin-containing cultivars lose their colour, instead of starting to synthesise pelargonidins, indicating the specificity of GDFR1-3 for the cyanidin pathway. This explains why petals of 'Ivory' are white, even when it has lost only one of the two enzymatically functional DFR forms, and shows that anthocyanin biosynthesis in gerbera is under more complex regulation than earlier thought.

Functional diversification of duplicated chalcone synthase genes in anthocyanin biosynthesis of Gerbera hybrida.

• Chalcone synthase (CHS) is the key enzyme in the first committed step of the flavonoid biosynthetic pathway and catalyzes the stepwise condensation of 4-coumaroyl-CoA and malonyl-CoA to naringenin chalcone. In plants, CHS is often encoded by a small family of genes that are temporally and spatially regulated. Our earlier studies have shown that GCHS4 is highly activated by ectopic expression of an MYB-type regulator GMYB10 in gerbera (Gerbera hybrida). • The tissue- and development-specific expression patterns of three gerbera CHS genes were examined. Virus-induced gene silencing (VIGS) was used to knock down GCHS1 and GCHS4 separately in gerbera inflorescences. • Our data show that GCHS4 is the only CHS encoding gene that is expressed in the cyanidin-pigmented vegetative tissues of gerbera cv Terraregina. GCHS3 expression is pronounced in the pappus bristles of the flowers. Expression of both GCHS1 and GCHS4 is high in the epidermal cells of gerbera petals, but only GCHS1 is contributing to flavonoid biosynthesis. • Gerbera contains a family of three CHS encoding genes showing different spatial and temporal regulation. GCHS4 expression in gerbera petals is regulated post-transcriptionally, at the level of either translation elongation or protein stability.

Detoxification and decolorization of a simulated textile dye mixture by phytoremediation using Petunia grandiflora and, Gailardia grandiflora: a plant-plant consortial strategy.

In vitro grown Petunia grandiflora and Gaillardia grandiflora plantlets showed 76 percent and 62 percent American Dye Manufacturers Institute value (color) removal from a simulated dyes mixture within 36h respectively whereas their consortium gave 94 percent decolorization. P. grandiflora, G. grandiflora and their consortium could reduce BOD by 44 percent, 31 percent and, 69 percent and COD by 58 percent, 37 percent and 73 percent respectively. Individually, root cells of P. grandiflora showed 74 and 24 percent induction in the activities of veratryl alcohol oxidase and laccase respectively; whereas G. grandiflora root cells showed 379 percent, 142 percent and 77 percent induction in the activities of tyrosinase, riboflavin reductase and lignin peroxidase respectively. In the consortium set, entirely a different enzymatic pattern was observed, where P. grandiflora root cells showed 231 percent, 12 percent and 65 percent induction in the activities of veratryl alcohol oxidase, laccase and 2, 6-dichlorophenol-indophenol reductase respectively, while G. grandiflora root cells gave 300 percent, 160 percent, 79 percent and 55 percent inductions in the activities of lignin peroxidase, riboflavin reductase, tyrosinase and laccase respectively. Because of the synergistic effect of the enzymes from both the plants, the consortium was found to be more effective for the degradation of dyes from the mixture. Preferential dye removal was confirmed by analyzing metabolites of treated dye mixture using UV-vis spectroscopy, FTIR and biotransformation was visualized using HPTLC. Metabolites formed after the degradation of dyes revealed the reduced cytogenotoxicity on Allium cepa roots cells when compared with untreated dye mixture solution. Phytotoxicity study exhibited the less toxic nature of the metabolites.

Cloning and sequence analysis of the Blumea balsamifera DC farnesyl diphosphate synthase gene.

Blumea balsamifera DC is a member of the Compositae family and is frequently used as traditional Chinese medicine. Blumea balsamifera is rich in monoterpenes, which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viral activities. Farnesyl diphosphate synthase (FPS) is a key enzyme in the biosynthetic pathway of terpenes, playing an important regulatory role in plant growth, such as resistance and secondary metabolism. Based on the conserved oligo amino acid residues of published FPS genes from other higher plant species, a cDNA sequence, designated BbFPS, was isolated from B. balsamifera DC using polymerase chain reaction. The clones were an average of 1.6 kb and contained an open reading frame that predicted a polypeptide of 342 amino acids with 89.07% identity to FPS from other plants. The deduced amino acid sequence was dominated by hydrophobic regions and contained 2 highly conserved DDxxD motifs that are essential for proper functioning of FPS. Phylogenetic analysis indicated that FPS grouped with other composite families. Prediction of secondary structure and subcellular localization suggested that alpha helices made up 70% of the amino acids of the sequence.

Functional characterization of geranyl/farnesyl diphosphate synthase in Wurfbainia villosa and Wurfbainia longiligularis.

Wurfbainia villosa and Wurfbainia longiligularis are the two primary plant sources of Fructus Amomi, a traditional Chinese medicine. Both plants are rich in volatile terpenoids, including monoterpenes and sesquiterpenes, which are the primary medicinal components of Fructus Amomi. The trans-isopentenyl diphosphate synthase (TIDS) gene family plays a key part in determining terpenoid diversity and accumulation. However, the TIDS gene family have not been identified in W. villosa and W. longiligularis. This study identified thirteen TIDS genes in W. villosa and eleven TIDS genes in W. longiligularis, which may have expanded through segmental replication events. Based on phylogenetic analysis and expression levels, eight candidate WvTIDSs and five WlTIDSs were selected for cloning. Functional characterization in vitro demonstrated that four homologous geranyl diphosphate synthases (GPPSs) (WvGPPS1, WvGPPS2, WlGPPS1, WlGPPS2) and two geranylgeranyl diphosphate synthases (GGPPSs) (WvGGPPS and WlGGPPS) were responsible for catalyzing the biosynthesis of geranyl diphosphate (GPP), whereas two farnesyl diphosphate synthases (FPPSs) (WvFPPS and WlFPPS) catalysed the biosynthesis of the farnesyl diphosphate (FPP). A comparison of six proteins with identified GPPS functions showed that WvGGPPS and WlGGPPS exhibited the highest activity levels. These findings indicate that homologous GPPS and GGPPS together promote the biosynthesis of GPP in W. villosa and W. longiligularis, thus providing sufficient precursors for the synthesis of monoterpenes and providing key genetic elements for Fructus Amomi variety improvement and molecular breeding.

Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis.

Lignins result from the oxidative polymerization of three hydroxycinnamyl (p-coumaryl, coniferyl, and sinapyl) alcohols in a reaction mediated by peroxidases. The most important of these is the cationic peroxidase from Zinnia elegans (ZePrx), an enzyme considered to be responsible for the last step of lignification in this plant. Bibliographical evidence indicates that the arabidopsis peroxidase 72 (AtPrx72), which is homolog to ZePrx, could have an important role in lignification. For this reason, we performed a bioinformatic, histochemical, photosynthetic, and phenotypical and lignin composition analysis of an arabidopsis knock-out mutant of AtPrx72 with the aim of characterizing the effects that occurred due to the absence of expression of this peroxidase from the aspects of plant physiology such as vascular development, lignification, and photosynthesis. In silico analyses indicated a high homology between AtPrx72 and ZePrx, cell wall localization and probably optimal levels of translation of AtPrx72. The histochemical study revealed a low content in syringyl units and a decrease in the amount of lignin in the atprx72 mutant plants compared to WT. The atprx72 mutant plants grew more slowly than WT plants, with both smaller rosette and principal stem, and with fewer branches and siliques than the WT plants. Lastly, chlorophyll a fluorescence revealed a significant decrease in ΦPSII and q L in atprx72 mutant plants that could be related to changes in carbon partitioning and/or utilization of redox equivalents in arabidopsis metabolism. The results suggest an important role of AtPrx72 in lignin biosynthesis. In addition, knock-out plants were able to respond and adapt to an insufficiency of lignification.

From Zinnia to Arabidopsis: approaching the involvement of peroxidases in lignification.

Zinnia elegans constitutes one of the most useful model systems for studying xylem differentiation, which simultaneously involves secondary cell wall synthesis, cell wall lignification, and programmed cell death. Likewise, the in vitro culture system of Z. elegans has been the best characterized as the differentiation of mesophyll cells into tracheary elements allows study of the biochemistry and physiology of xylogenesis free from the complexity that heterogeneous plant tissues impose. Moreover, Z. elegans has emerged as an excellent plant model to study the involvement of peroxidases in cell wall lignification. This is due to the simplicity and duality of the lignification pattern shown by the stems and hypocotyls, and to the basic nature of the peroxidase isoenzyme. This protein is expressed not only in hypocotyls and stems but also in mesophyll cells transdifferentiating into tracheary elements. Therefore, not only does this peroxidase fulfil all the catalytic requirements to be involved in lignification overcoming all restrictions imposed by the polymerization step, but also its expression is inherent in lignification. In fact, its basic nature is not exceptional since basic peroxidases are differentially expressed during lignification in other model systems, showing unusual and unique biochemical properties such as oxidation of syringyl moieties. This review focuses on the experiments which led to a better understanding of the lignification process in Zinnia, starting with the basic knowledge about the lignin pattern in this plant, how lignification takes place, and how a sole basic peroxidase with unusual catalytic properties is involved and regulated by hormones, H2O2, and nitric oxide.

Isolation and functional analysis of a homolog of flavonoid 3',5'-hydroxylase gene from Pericallis × hybrida.

As the key enzyme in the biosynthesis of blue flower color pigments, flavonoid 3',5'-hydroxylase (F3'5'H) can catalyze the conversion of its major substrates, 2-S naringenin and dihydrokaempferol, into 3',4',5'-hydroxylated pentahydroxyflavanone and dihydromyricetin, respectively. Unlike other F3'5'Hs belonging to the CYP75A subfamily, Asteraceae-specific F3'5'Hs belong to the CYP75B subfamily. Furthermore, cineraria F3'5'H expressed in yeast exhibited not only F3'H (flavonoid 3'-hydroxylase) activity but also F3'5'H activity in vitro. In this study, Southern blotting showed that there was only one copy of a homolog of the F3'5'H gene PCFH in the Pericallis × hybrida genome. This gene could be detected by Northern blot in the primary developmental stages of ligulate florets of the purple- and blue-flowered cultivars, and its transcripts also accumulated in the leaves. Heterologous expression of PCFH could produce new delphinidin derivatives in the corollas of transgenic tobacco plants, increased the content of cyanidin derivatives and lead to the blue- and red-shifting of flower color in T0 generation plants. These results indicate that cineraria F3'5'H exhibited both F3'5'H- and F3'H-activity in vivo. The types and contents of anthocyanins and flower color phenotypes of the T1 generation were similar to those of T0 generation plants. PCFH exhibited stable inheritance and normal functions between generations. This study supplies new evidence to understand Asteraceae-specific F3'5'Hs and provides important references for the further study of molecular breeding of blue-flowered chrysanthemums using the PCFH gene.

Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism.

BACKGROUND: Chrysanthemyl diphosphate synthase (CDS) is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS) gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. RESULTS: Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. CONCLUSION: Positive selection associated with gene duplication played a major role in the evolution of CDS.

The promoter region of the Zinnia elegans basic peroxidase isoenzyme gene contains cis-elements responsive to nitric oxide and hydrogen peroxide.

NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.

Biochemical conservation and evolution of germacrene A oxidase in asteraceae.

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes approximately 8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.

Ethylene stimulates tracheary element differentiation in Zinnia elegans cell cultures.

The exact role of ethylene in xylogenesis remains unclear, but the Zinnia elegans cell culture system provides an excellent model with which to study its role during the differentiation of tracheary elements (TEs) in vitro. Here, we analysed ethylene homeostasis and function during Z. elegans TE differentiation using biochemical, molecular and pharmacological methods. Ethylene evolution was confined to specific stages of TE differentiation. It was found to peak at the time of TE maturation and to correlate with the activity of the ethylene biosynthetic 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The ethylene precursor ACC was exported and accumulated to high concentrations in the extracellular medium, which also displayed a high capacity to convert ACC into ethylene. The effects of adding inhibitors of the ethylene biosynthetic ACC synthase and ACC oxidase enzymes to the TE cultures demonstrated for the first time strict dependence of TE differentiation on ethylene biosynthesis and a stimulatory effect of ethylene on the rate of TE differentiation. In a whole-plant context, our results suggest that ethylene synthesis occurs in the apoplast of the xylem elements and that ethylene participates, in a paracrine manner, in the control of the cambial stem cell pool size during secondary xylem formation.

Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase.

Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between beta3-2 and beta3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.