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We conducted biochemical and physiological experiments to investigate the mode of action of tiafenacil (Terrad'or™), a new protoporphyrinogen IX oxidase (PPO)-inhibiting pyrimidinedione herbicide. Analysis of the half-maximal inhibitory concentration (IC 50 ) against recombinant PPO enzymes from various plant species, including amaranth (Amaranthus tuberculatus), soybean (Glycine max), arabidopsis (Arabidopsis thaliana), and rapeseed (Brassica napus), showed that tiafenacil had an IC 50 of 22 to 28 nM, similar to the pyrimidinedione herbicides butafenacil and saflufenacil and the N-phenylphthalimide herbicide flumioxazin. By contrast, tiafenacil exhibited 3- to 134-fold lower IC 50 values than the diphenyl ether herbicides fomesafen, oxyfluorfen, and acifluorfen. Tiafenacil is non-selective and is herbicidal to both dicots and monocots, such as the weeds velvetleaf (Abutilon theophrasti), amaranth, and barnyardgrass (Echinochloa crus-galli) as well as the crops soybean, rapeseed, rice (Oryza sativa), and maize (Zea mays) at concentrations ranging from 1 to 50 μM. Treatment of plant tissue with tiafenacil in darkness resulted in the accumulation of protoporphyrin IX. Subsequent exposure to light increased the content of malondialdehyde and significantly decreased the Fv/Fm values of chlorophyll fluorescence. The results suggest that tiafenacil is a new PPO-inhibiting pyrimidinedione herbicide., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Enzymes of the thiolase superfamily catalyze the formation of carbon-carbon bond via the Claisen condensation reaction. Thiolases catalyze the reversible non-decarboxylative condensation of acetoacetyl-CoA from two molecules of acetyl-CoA, and possess a conserved Cys-His catalytic diad. Elongation enzymes (beta-ketoacyl-acyl carrier protein synthase (KAS) I and KAS II and the condensing domain of polyketide synthase) have invariant Cys and two His residues (CHH triad), while a Cys-His-Asn (CHN) triad is found in initiation enzymes (KAS III, 3-ketoacyl-CoA synthase (KCS) and the chalcone synthase (CHS) family). These enzymes all catalyze decarboxylative condensation reactions. 3-Hydroxyl-3-methylglutaryl-CoA synthase (HMGS) also contains the CHN triad, although it catalyzes a non-decarboxylative condensation. That the enzymes of the thiolase superfamily share overall similarity in protein structure and function suggested a common evolutionary origin. All thiolases were found to have, in addition to the Cys-His diad, either Asn or His (thus C(N/H)H) at a position corresponding to the His in the CHH and CHN triads. In our phylogenetic analyses, the thiolase superfamily was divided into four main clusters according to active site architecture. During the functional divergence of the superfamily, the active architecture was suggested to evolve from the C(H)H in archaeal thiolases to the C(N/H)H in non-archaeal thiolases, and subsequently to the CHH in the elongation enzymes and the CHN in the initiation enzymes. Based on these observations and available biochemical and structural evidences, a plausible evolutionary history for the thiolase superfamily is proposed that includes the emergence of decarboxylative condensing enzymes accompanied by a recruitment of the His in the CHH and CHN triads for a catalytic role during decarboxylative condensation. In addition, phylogenetic analysis of the plant CHS family showed separate clustering of CHS and non-CHS members of the family with a few exceptions, suggesting repeated gene birth-and-death and re-invention of non-CHS functions throughout the evolution of angiosperms. Based on these observations, predictions on the enzymatic functions are made for several members of the CHS family whose functions are yet to be characterized. Further, a moss CHS-like enzyme that is functionally similar to a cyanobacterial enzyme was identified as the most recent common ancestor to the plant CHS family.

An apple starch-branching enzyme SbeI gene (GenBank Accession No. DQ115404) has been isolated, cloned, and sequenced. The SbeI is a single copy gene in the apple genome, consisting of 14 exons and 13 introns, and covering 6075bp. As detected by RT-PCR, the apple SbeI is expressed at very low levels during early stages of fruit development; while, the highest levels of mRNA transcripts are observed at approximately 44 days post-pollination. Besides fruits, the apple SbeI is also expressed in buds and flowers, and very weakly in leaves. The genomic structure of SbeI in apple is strikingly similar to those reported so far in grasses (Poaceae), with exons 4 through 13 being of identical lengths in both apple and grasses. Moreover, structure similarities in exon lengths have also been detected in SbeII genes of both grasses and eudicots. These findings prompted the investigation of the evolutionary process of the Sbe gene family in angiosperms. A total of 26 Sbe sequences, representing an array of monocots and eudicots, are investigated in this study. Phylogenetic analysis has suggested that Sbe genes have duplicated into SbeI and SbeII prior to the divergence of moncots from eudicots. The SbeII gene is further duplicated into SbeIIa and SbeIIb prior to the radiation of grasses; however, it is not yet clear whether this duplication event has occurred before or after the radiation of the eudicots.

NADP-dependent isocitrate dehydrogenase is a low-copy nuclear gene family. We have sequenced two regions from an idh gene (idhB) near the 3' terminal end. The first fragment encodes 4 exons and 3 introns and is between approximately 600 and 950 bp in length. The second fragment includes three additional exons and introns and is between approximately 1200 and 1500 bp in length. The phylogenetic utility of the two sequence regions was evaluated in Polemoniaceae with a focus on Saltugilia, an incipient species complex that lacks phylogenetic resolution among these same taxa based on nuclear ribosomal ITS and chloroplast trnL. Multiple sequences from several individuals, multiple individuals from several populations, and multiple populations from all Saltugilia species were sampled to evaluate the taxonomic level at which idhB was useful as a phylogenetic marker in this clade. Phylogenies based on idhB sequences were compared with topological resolution and clade composition in ITS and trnL phylogenies. Phylogenies based on idhB and idhB in combination with ITS and trnL are better resolved than any other phylogenies for Saltugilia published to date, and character evolution within Saltugilia is explored.

S-RNase-based gametophytic self-incompatibility appears to be the most phylogenetically widespread form of self-incompatibility found in the angiosperms, having been reported in the Solanaceae, Scrophulariaceae, and Rosaceae. This intraspecific breeding barrier is controlled by a single genetic locus termed S. Rejection of self-pollen has been shown to be mediated in the pistil by a highly polymorphic series of ribonucleases, but as yet the pollen component of this recognition system has not been identified. Here we review our present knowledge concerning the structure, functions, and evolution of S-RNases and the S-loci in which they reside. In addition we present two new phylogenetic analyses of S-RNases which suggest that (1). sequence variability between S-alleles is spread across the whole gene and is not as clustered as is generally believed and (2). there is evidence of recombination and/or diversifying selection in two distinct regions of S-RNases. The implications of these findings are discussed.

We used sequences of nrDNA ITS and chloroplast gene matK to evaluate the monophyly of Empetrum and Corema and to examine phylogenetic relationships of the Empetraceae. Sequences of these two DNA markers were obtained for 11 plant samples, representing species of Empetrum from both the Southern and Northern Hemispheres, species and subspecies of Corema, and the monotypic Ceratiola. Sequences of four species of Rhododendron were used for rooting purposes. Our results show that species of Empetrum form a clade sister to the clade containing both Corema and Ceratiola. These two clades are strongly supported in both the matK and ITS trees, suggesting that Ceratiola is more closely related to Corema than to Empetrum, and is not of a hybrid origin between the ancestors of the latter two genera. In the matK tree, Corema conradii is more closely related to Ceratiola than to Corema album and C. album subsp. azoricum, whereas in the ITS tree, Ceratiola is allied with Corema album and C. album subsp. azoricum. This suggests that C. conradii might be a hybrid between ancestral populations of Ceratiola and C. album. The monophyly of Empetrum rejects the hypothesis of its independent origin in the two Hemispheres. Our trees also suggest the fact that the modern amphitropical distribution of Empetrum is the result of long distance dispersal, not of the vicarious events., (Copyright 2002 Elsevier Science (USA))

We sampled the 5' end of the granule-bound starch synthase gene (GBSSI or waxy) in Rosaceae, sequencing 108 clones from 18 species in 14 genera representing all four subfamilies (Amygdaloideae, Maloideae, Rosoideae, and Spiraeoideae), as well as four clones from Rhamnus catharticus (Rhamnaceae). This is the first phylogenetic study to use the 5' portion of this nuclear gene. Parsimony and maximum-likelihood analyses of 941 bases from seven complete and two partial exons demonstrate the presence of two loci (GBSSI-1 and GBSSI-2) in the Rosaceae. Southern hybridization analyses with locus-specific probes confirm that all four Rosaceae subfamilies have at least two GBSSI loci, even though only one locus has been reported in all previously studied diploid flowering plants. Phylogenetic analyses also identify four clades representing four loci in the Maloideae. Phylogenetic relationships inferred from GBSSI sequences are largely compatible with those from chloroplast (cpDNA: ndhF, rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) DNA. Large clades are marked by significant intron variation: a long first intron plus no sixth intron in Maloideae GBSSI-1, a long fourth intron in Rosoideae GBSSI-1, and a GT to GC mutation in the 5' splice site of the fourth intron in all GBSSI-2 sequences. Our data do not support the long-held hypothesis that Maloideae originated from an ancient hybridization between amygdaloid and spiraeoid ancestors. Instead, Spiraeoideae genera (Kageneckia and Vauquelinia) are their closest relatives in all four GBSSI clades., (Copyright 2000 Academic Press.)

A phylogenetic study of Asteridae sensu lato was conducted based on chloroplast ndhF gene sequences for 116 ingroup and 13 outgroup species. Prior molecular studies based on rbcL sequences identified terminal groups corresponding to families, but were unable to resolve relationships among them. These results are largely consistent with earlier rbcL studies, but provide much greater resolution and stronger bootstrap support throughout the tree. The parsimony analysis found eight equally parsimonious trees, all of which recognize four major clades with the following relationship: (Cornales (Ericales (Euasterids I, Euasterids II))). Euasterids I includes (Garryales ((Solanales, Boraginaceae) (Gentianales, Lamiales))), although with weak support for relationships among the named clades. Euasterids II includes (Aquifoliales (Asterales (Apiales, Dipsacales))) with strong support for these relationships. Relationships within Ericales are weakly supported and merit further attention., (Copyright 2000 Academic Press.)