Comparison of ALS functionality and plant growth in ALS-inhibitor susceptible and resistant Myosoton aquaticum L.

Herbicide target-site resistance mutations may cause pleiotropic effects on plant ecology and physiology. The effect of several known (Pro197Ser, Pro197Leu Pro197Ala, and Pro197Glu) target-site resistance mutations of the ALS gene on both ALS functionality and plant vegetative growth of weed Myosoton aquaticum L. (water chickweed) have been investigated here. The enzyme kinetics of ALS from four purified water chickweed populations that each homozygous for the specific target-site resistance-endowing mutations were characterized and the effect of these mutations on plant growth was assessed via relative growth rate (RGR) analysis. Plants homozygous for Pro197Ser and Pro197Leu exhibited higher extractable ALS activity than susceptible (S) plants, while all ALS mutations with no negative change in ALS kinetics. The Pro197Leu mutation increased ALS sensitivity to isoleucine and valine, and Pro197Glu mutation slightly increased ALS sensitivity to isoleucine. RGR results indicated that none of these ALS resistance mutations impose negative pleiotropic effects on relative growth rate. However, resistant (R) seeds had a lowed germination rate than S seeds. This study provides baseline information on ALS functionality and plant growth characteristics associated with ALS inhibitor resistance-endowing mutations in water chickweed.

[Cloning and expression analysis on full length CDS of zeaxanthin epoxidase gene in Pseudostellaria heterophylla].

Zeaxanthin epoxidase plays an important role in indirect pathway of plant abscisic acid biosynthesis. According to the data of Pseudostellaria heterophylla transcriptome, zeaxanthin epoxidase gene was isolated and named as PhZEP. The results of bioinformatics analysis showed that the coding sequence of PhZEP was 1 263 bp long and encoded 420 amino acids. The putative protein molecular weight was 47.34 kDa and its theoretical isoelectric point was 6.64. The characteristic structure domains were predicted, including binding site of lipoprotein and flavoprotein monooxyenase. A signal peptide was discovered at the N-terminal of amino acids. The Real-time PCR revealed that PhZEP had a higher expression level in leaves than other tissues of P.heterophylla. Highly expressed PhZEP was also observed at 10 d and 40 d tuberous root after flowering. PhZEP presented a different expression after treatment with ABA, fluridone and ABA +fluridone compared to the control. The expression of PhZEP in tuberous root after ABA treatment was close to that in control group, while PhZEP showed significant up-regulation in the fluridone treatment group. In this study, the PhZEP gene from P. heterophylla was cloned and this result has important significance for its functional identification. This research provides a basis for the further analysis on functional mechanism of ABA during development of P. heterophylla.

[Cloning and expression analysis of four carotenoid cleavage dioxygenases genes from Pseudostellaria heterophylla].

To investigate the molecular mechanism of quality formation of Pseudostellaria heterophylla, the carotenoid cleavage dioxygenases (CCDs) genes were cloned from the transcriptome database of P. heterophylla, and analyzed them with bioinformatics analysis and expression analysis. The sequence length of four new gene were 1 617, 1 461, 1 746, 1 875 bp, and subsequently, named as PhCCD1,PhNCED2,PhNCED3 and PhCCD4 according to its genetic relationship with Arabidopsis thaliana. The sequence analysis showed that four new gene were all containing REP65 domains and binding sites of ferrous ion, such as histidine, glutamates and aspartates. Analysis phylogeny showed that PhNCED2 and PhNCED3 were the cluster of NCEDs, PhCCD1 and PhCCD4 were the cluster of CCDs. In addition, PhCCD1 and AtCDD1 of Arabidopsis thaliana, PhCCD4 and AtCCD4 of A. thaliana,PhNCED2, PhNCED3 and AtNCED3 of A. thaliana have high similarities. Analysis of real-time fluorescence quantitative showed that PhNCED2 and PhNCED3 were expressed mainly in underground part, the expression quantity of PhNCED2 reached the highest in fibrous root, PhNCED3 keeps higher in phloem and xylem, it may be the key enzymes of ABA biosynthesis genes. Moreover,PhCCD1 and PhCCD4 were expressed mainly in aerial part,the expression quantity of PhCCD1 reached the highest in leaf,PhCCD4 keeps higher in stem and leaf.It may be involved in the biosynthesis of carotenoids for P. heterophylla. The study obtained CDDs gene of P. heterophylla for the first time,this would lay the foundation of developing the response mechanism of P. heterophylla about external stress further,and then exploring the biological approach of quality formation in P. heterophylla.

[Cloning and bioinformatics analysis of abscisic acid 8'-hydroxylase from Pseudostellariae Radix].

Abscisic acid 8'-hydroxylase was one of key enzymes genes in the metabolism of abscisic acid (ABA). Seven menbers of abscisic acid 8'-hydroxylase were identified from Pseudostellaria heterophylla transcriptome sequencing results by using sequence homology. The expression profiles of these genes were analyzed by transcriptome data. The coding sequence of ABA8ox1 was cloned and analyzed by informational technology. The full-length cDNA of ABA8ox1 was 1 401 bp,with 480 encoded amino acids. The predicated isoelectric point (pI) and relative molecular mass (MW) were 8.55 and 53 kDa,respectively. Transmembrane structure analysis showed that there were 21 amino acids in-side and 445 amino acids out-side. High level of transcripts can detect in bark of root and fibrous root. Multi-alignment and phylogenetic analysis both show that ABA8ox1 had a high similarity with the CYP707As from other plants,especially with AtCYP707A1 and AtCYP707A3 in Arabidopsis thaliana. These results lay a foundation for molecular mechanism of tuberous root expanding and response to adversity stress.

A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

Molecular and functional evolution of class I chitinases for plant carnivory in the caryophyllales.

Proteins produced by the large and diverse chitinase gene family are involved in the hydrolyzation of glycosidic bonds in chitin, a polymer of N-acetylglucosamines. In flowering plants, class I chitinases are important pathogenesis-related proteins, functioning in the determent of herbivory and pathogen attack by acting on insect exoskeletons and fungal cell walls. Within the carnivorous plants, two subclasses of class I chitinases have been identified to play a role in the digestion of prey. Members of these two subclasses, depending on the presence or absence of a C-terminal extension, can be secreted from specialized digestive glands found within the morphologically diverse traps that develop from carnivorous plant leaves. The degree of homology among carnivorous plant class I chitinases and the method by which these enzymes have been adapted for the carnivorous habit has yet to be elucidated. This study focuses on understanding the evolution of carnivory and chitinase genes in one of the major groups of plants that has evolved the carnivorous habit: the Caryophyllales. We recover novel class I chitinase homologs from species of genera Ancistrocladus, Dionaea, Drosera, Nepenthes, and Triphyophyllum, while also confirming the presence of two subclasses of class I chitinases based upon sequence homology and phylogenetic affinity to class I chitinases available from sequenced angiosperm genomes. We further detect residues under positive selection and reveal substitutions specific to carnivorous plant class I chitinases. These substitutions may confer functional differences as indicated by protein structure homology modeling.

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata.

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [ß-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.

A new type 1 ribosome-inactivating protein from the seeds of Gypsophila elegans M.Bieb.

Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.

[Cloning and characterization of cDNA encoding Psammosilene tunicoides squalene synthase].

The total triterpene saponins of Psammosilene tunicoides have significant pharmacologic activity. Psammosilene tunicoides squalene synthase (PSS) is a gateway enzyme to regulate the biosynthesis of total triterpene saponins extracted from the root of Psammosilene tunicoides which is an endangered species. In this paper, cDNA encoding of PSS was cloned by the degenerate primer PCR and rapid-amplification of cDNA ends (RACE). The full-length of cDNA of PSS is 1663 bp, with an open reading frame (ORF) of 1 245 bp, encoding 414 amino acid polypeptide (calculated molecular mass, 47.69 kDa), 5'UTR (untranslated region) and 3'UTR are 260 bp and 158 bp, respectively. The deduced amino acid sequence of PSS has higher homology with the known squalene synthases of several species such as Panax notoginseng (83%), Panax ginseng (82%) and Glycyrrhiza glabra (82%) than that with Schizosacharomyces pombe (35%), Candida albicans (39%) and Homo sapiens (47%). The characterization of PSS was done by a series of methods, such as prokaryotic expression, the activity of enzyme in vitro, capillary gas chromatography (GC) and capillary gas chromatography mass spectrometry (GC-MS). The results showed that the cell-free extract of E. coli transformed with the recombinant plasmid can effectively convert farnesyl diphosphate into squalene in vitro. GenBank accession number is EF585250. Our research provided important base for the study of Psammosilene tunicoides secondary metabolism and metabolic engineering.

[Studies on strain characteristics of Pseudostellaria heterophylla varieties].

OBJECTIVE: To characterize the different varieties of Pseudostellaria heterophylla during cultivation. METHOD: Using systematic selection in the main productive areas, the techniques of random design, all varieties were observed for 3 years. RESULT: The biological and 425 productive characteristics of P. heterophylla var. macrophylla, P. heterophylla var. Foliolum, and P. heterophylla var. anvense were significantly different (P < 0.01). There were also differences in ecological adaptability, plant characteristics, pollen granule, chromosomes, and isoenzyme of the three cultivars. CONCLUSION: The strain types of P. heterophylla was denominated for the first time. The characteristics and productivity index system of P. heterophylla varieties were determined.

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex.

BACKGROUND: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. RESULTS: To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. CONCLUSIONS: All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

Unexpectedly high genetic variation in large unisexual clumps of the subdioecious plant Honckenya peploides (Caryophyllaceae).

Honckenya peploides is a subdioecious dune plant that reproduces both sexually and by clonal growth. In northwest Spain this species was found to exhibit an extreme spatial segregation of the sexes, and our objective was to investigate genetic variation in unisexual clumps. Genetic variation was studied in six unisexual clumps of H. peploides, three of them exclusively composed of males and three exclusively female. In total, 193 samples were analysed using isozyme analysis and 80 samples were analysed using two AFLP primer combinations. Both techniques revealed considerably high genetic diversity (average proportion of distinguishable genotypes: 0.22 for isozymes and 0.36 for AFLP; average Simpson's D: 0.65 for isozymes and 0.68 for AFLP). Our results show that, in spite of clonal growth, each unisexual clump consists of different genotypes. Genetic diversity within clumps is similar for both sexual morphs. Reasons for unisexuality of the clumps are discussed.

Dihydroflavonol 4-reductase cDNA from non-anthocyanin-producing species in the Caryophyllales.

Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plant. Although anthocyanins are widely distributed in higher plants as flower and fruit pigments, betacyanins have replaced anthocyanins in the Caryophyllales. We isolated cDNAs encoding dihydroflavonol 4-reductase (DFR), which is the first enzyme committed to anthocyanin biosynthesis in the flavonoid pathway, from Spinacia oleracea and Phytolacca americana, plants that belong to the Caryophyllales. The deduced amino acid sequence of Spinacia DFR and Phytolacca DFR revealed a high degree of homology with DFRs of anthocyanin-producing plants. The DFR of carnation, an exception in the Caryophyllales that synthesizes anthocyanin, showed the highest level of identity. In the phylogenetic tree, Spinacia DFR and Phytolacca DFR clustered with the DFRs of anthocyanin-synthesizing dicots. Recombinant Spinacia and Phytolacca DFRs expressed in Escherichia coli convert dihydroflavonol to leucoanthocyanidin. The expression and function of DFR in spinach and pokeweed are discussed in relation to the molecular evolution of red pigment biosynthesis in higher plants.