Heterologous expression and structural characterisation of a pyrazinone natural product assembly line.

Through a number of strategies nonribosomal peptide assembly lines give rise to a metabolic diversity not possible by ribosomal synthesis. One distinction within nonribosomal assembly is that products are elaborated on an enzyme-tethered substrate, and their release is enzyme catalysed. Reductive release by NAD(P)H-dependent catalysts is one observed nonribosomal termination and release strategy. Here we probed the selectivity of a terminal reductase domain by using a full-length heterologously expressed nonribosomal peptide synthetase for the dipeptide aureusimine and were able to generate 17 new analogues. Further, we generated an X-ray structure of aureusimine terminal reductase to gain insight into the structural details associated with this enzymatic domain.

Conserved, unstructured regions in Pseudomonas aeruginosa PilO are important for type IVa pilus function.

Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilOΔ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous ß-strand in the second αßß motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.

Cytokinin autonomy in tissue cultures of phaseolus: a genotype-specific and heritable trait.

Intra- and interspecific differences in cytokinin requirement were detected in callus cultures of Phaseolus vulgaris L. and P. lunatus L. Of the ten genotypes of P. vulgaris tested in the present study, one required cytokinin for callus growth, six exhibited some to moderate growth on cytokinin-free medium, and the remaining three grew uniformly in the absence of cytokinin. In contrast, six of the P. lunatus genotypes were strictly cytokinin-dependent, while four genotypes displayed irregular amount of callus growth on cytokinin-free medium. The genotype-specific behavior of Phaseolus callus tissues was independent of the tissue of origin and the time in culture. The inheritance of the cytokinin requirement of Phaseolus tissue cultures was studied in hybrid tissues resulting from crosses between a strictly cytokinin-dependent genotype (P.I. 200960) and two independent genotypes (cv. G 50 and P.I. 286303) of P. vulgaris. Fresh weights of hybrid tissues on cytokinin-free medium were intermediate between and significantly different from the parental tissues. No differences were found between reciprocal hybrids. These results suggest that cytokinin autonomy in tissue cultures of P. vulgaris is a genetic trait under nuclear control. Both parental and intermediate phenotypes were recovered in the F(2) progeny. The frequency distribution of cytokinin-dependent progeny in F(2) and backcross populations indicates that the cytokinin requirement of P. vulgaris callus tissue may be regulated by one set of alleles.

Partial Purification of a cis-trans-Isomerase of Zeatin from Immature Seed of Phaseolus vulgaris L.

Investigation of the conversion of exogenous cis-zeatin to trans-zeatin in immature seeds of Phaseolus vulgaris L. led to the isolation of a cis-trans-isomerase from the endosperm. The enzyme was purified more than 2000-fold by chromatography on a series of fast protein liquid chromatography (anion exchange, gel filtration, and hydrophobic interaction) and concanavalin A columns. The enzymic reaction favors conversion from the cis to the trans form and requires flavin, light, and dithiothreitol. cis-Zeatin riboside is also a substrate for the enzyme. Retention on the concanavalin A column indicated that the enzyme is a glycoprotein. The enzyme was stable for at least 8 weeks when stored at -80[deg] C. The occurrence of cis-trans-isomerization suggests that cis-zeatin and cis-zeatin riboside formed by tRNA degradation could be precursors of biologically active cytokinins.

Monosomics in common bean, Phaseolus vulgaris.

Two monosomics of Phaseolus vulgaris (2n = 22) were found among selfed progeny of plants treated with colchicine. The monosomic chromosomes involved were identified as chromosomes H and J according to the previously suggested Giemsa karyotype. Both monosomic plants had slower growth rate and smaller size as compared with their respective euploid sibs. However, no apparent morphological characteristics distinguished the two monosomics. The frequencies of transmission through selfing of monosomics H and J were 9% and 10 % respectively.

Metabolism of C-zeatin in phaseolus embryos : occurrence of o-xylosyldihydrozeatin and its ribonucleoside.

The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5'-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.

Differential cytokinin structure-activity relationships in phaseolus.

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N(6) side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N(6)-(Delta(2)-isopentenyl)adenine and N(6)-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N(6)-isopentyladenine was 100-fold more active than N(6)-(Delta(2)-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.

Interspecific hybridization of phaseolus vulgaris with P. lunatus and P. acutifolius.

The influence of genotypic combinations on the growth of hybrid embryos between Phaseolus vulgaris and P. lunatus, and between P. vulgaris and P. acutifolius was examined. All embryos obtained from P. vulgaris × P. lunatus crosses developed only to a stage which appears to be comparable to the pre-heart-shape stage of selfed embryos. Reciprocal crosses were attempted, but pods abscised at a very early stage. Embryos derived from P. vulgaris × P. acutifolius and reciprocal crosses attained the cotyledon stage although no mature seeds were formed. A distinct characteristic of these embryos was the uneven development of the two cotyledons. The rate of growth and final size of these hybrid embryos seemed to be influenced by the genotypes of both parents.Immature embryos were cultured on defined medium and the effects of glutamine and gibberellin (GA3) were examined. Glutamine was effective in increasing the survival rate; gibberellin had no apparent effect. Plants derived from cultured embryos of P. vulgaris × P. lunatus, P. vulgaris × P. acutifolius and P. acutifolius × P. vulgaris were obtained.

Fertilization and early embryo development in reciprocal interspecific crosses of Phaseolus.

Fertilization and early embryo and endosperm development were examined in Phaseolus vulgaris x P. acutifolius, P. vulgaris x P. lunatus crosses and their reciprocals. The number and length of pollen tubes were not different between selfings and interspecific crosses. Fertilization was completed in all matings and the time of fertilization was maternally dependent which may reflect the degree of maturation of embryo sacs at pollination. A large difference between reciprocal crosses was found in the time of endosperm and embryo division in relation to the time of fertilization. When P. vulgaris was the female parent and P. acutifolius the male parent, endosperm division occurred at the same time as in P. vulgaris upon selfing, while in P. vulgaris x P. lunatus crosses the time of endosperm division was intermediate as compared with the two parents. The time lapse between fertilization and endosperm and embryo division in P. acutifolius x P. vulgaris crosses was longer than in either parent upon selfing. In P. lunatus x P. vulgaris crosses, endosperm division occurred in only 7-12% of the ovules at 72 hours after pollination. Embryo development in these ovules was limited to the four cell stage although the endosperm was at the free nuclei stage. The severe delay in embryo and endosperm divisions may be the major cause of early pod abscission in P. lunatus x P. vulgaris crosses.

Plantlet regeneration from cultured leaves of Cydonia oblonga L. (quince).

Adventitious shoots of Cydonia oblonga Quince A were obtained from leaves cultured on MS-N6 medium containing thidiazuron (TDZ) and α-naphthaleneacetic acid (NAA). The frequency of regeneration was high (78% of the cultured leaves with 3.2 shoots per regenerating leaf) at 32 µM TDZ plus 0.3 µM NAA on young leaves obtained from micropropagated shoots. Shoots were rooted by culturing them first on medium containing 5 µM NAA for one week and then on auxinfree medium for four weeks. The regeneration protocol may be useful for selection of somaclonal variants with increased tolerance to low Fe and for transformation mediated by Agrobacterium.

Zeatin Metabolism in Fruits of Phaseolus: Comparison between Embryos, Seedcoat, and Pod Tissues.

Zeatin metabolites were isolated from seedcoats and pod tissues of Phaseolus vulgaris and P. lunatus. The differences observed previously between P. vulgaris and P. lunatus embryos, i.e. the formation of O-ribosyl derivatives in the former and O-glucosyl derivatives in the latter, could also be detected in seedcoats, although the levels of these metabolites were much lower and there was a concomitant increase of breakdown products (adenine, adenosine and AMP). Inner pod wall tissues of both genotypes metabolized zeatin at a slow rate and the major metabolite was the mononucleotide of zeatin. The array of metabolites recovered was not influenced by the extraction method (cold ethanol or modified Bieleski solution).

Somatic embryogenesis and shoot organogenesis from interspecific hybrid embryos of Vigna glabrescens and V. radiata.

Somatic embryogenesis occurred on cotyledons of morphologically abnormal embryos derived from Vigna glabrescens x V. radiata crosses and cultured on Murashige and Skoog (MS) medium without growth regulators. The frequency of 15-17 day old embryos that gave rise to somatic embryos increased from 8% to 29% by application a mixture of 100 mg/l gibberelllc acid, 25 mg/l α-naphthaleneacetic acid (NAA) and 5 mg/l kinetin daily to the pedicels of the developing pods. However, only callus formed on immature hybrid embryos of the reciprocal cross. These callus tissues occasionally gave rise to shoots via organogenesis when transferred to MS medium with 2 mg/l N(6)-benzyladenine and 0.05 mg/l NAA. Treatment of pods with growth regulators did not influence the frequency of organogenic callus. Selfed embryos of the parents did not form somatic embryos in culture, nor did callus derived from the selfed embryos produce shoots. Thus, the ability to redifferentiate appears to be associated with interspecific hybridity.

RFLP analysis of preferential transmission in interspecific hybrids of Phaseolus vulgaris and P. coccineus.

The inheritance of RFLP markers in interspecific hybrids of Phaseolus vulgaris and P. coccineus was analyzed. Of 280 cDNA probes used, 70%-85% revealed polymorphism between species while intraspecific RFLP ranged from 8% to 37%. Segregation of 63 clearly scorable markers was examined in 177 P. vulgaris x P. coccineus F2's maintained as callus. Preferential transmission of the P. vulgaris alleles was observed for 24 of the 28 loci exhibiting non-Mendelian ratios. Although the segregation ratios at 17 loci fit gametic selection, also other factors such as nuclear-cytoplasmic or embryo-endosperm interactions may be involved. In the reciprocal F2, a relatively high frequency of maternal alleles was recovered for several loci, while the paternal allele was favored at others. The cDNA clone detecting the most extreme segregation, with no P. coccineus type detected among 165 P. vulgaris x P. coccineus F2 progeny, showed high homology to histone H2A genes. The markers were mapped to nine linkage groups. Aggregation of markers with preferential maternal transmission was observed, which could be due to selection of individual chromosomes, although false linkage detection cannot be excluded. The results obtained with RFLPs may explain the skewed distribution of phenotypic traits following interspecific hybridization.

Protein processing and auxin response in transgenic tobacco harboring a putative cDNA of zeatin O-xylosyltransferase from Phaseolus vulgaris.

Zeatin is rapidly metabolized to O-xylosylzeatin in Phaseolus vulgaris seeds. The zeatin O-xylosyltransferase mediating this conversion, a 50 kDa protein, occurs mainly in the endosperm, both in the cytoplasm and the nuclei. A monoclonal antibody specific to the enzyme was used to isolate cDNAs from an expression library derived from P. vulgaris seeds. Two highly homologous, full-length cDNAs were isolated. The ORFs encode proteins of 69 and 67 kDa, respectively, with 90% homology at the amino acid level. cDNA-encoded protein obtained from in vitro transcription/translation was processed to protein of 50 kDa by bean endosperm extract. Transgenic tobacco plants harboring the larger ORF under the control of the CaMV35S promoter were more sensitive to the auxin NAA than control plants. The symptoms included leaf chlorosis, restriction of root elongation, and eventual cessation of growth. The antigenic preprotein was processed, and labeled zeatin was converted to O-xylosylzeatin in transgenic plants grown on NAA-containing medium. Analyses of independently transformed families indicated that the presence of the transgene coincided with the increased auxin sensitivity and protein processing correlated with the manifestation of auxin-induced damage. These results suggest that posttranslational processing regulates enzyme activity, and offer the possibility that cytokinin-auxin balance may be affected by stimulation of cytokinin metabolic enzyme activity by auxin.

A gene encoding the cytokinin enzyme zeatin O-xylosyltransferase of Phaseolus vulgaris.

Zeatin is the most active and ubiquitous form of the naturally occurring cytokinins. Glycosyl conjugates of zeatin are found in many plant tissues and are considered important for storage and protection against degradative enzymes. Two enzymes catalyzing the formation of O-glycosyl derivatives of zeatin have been characterized, O-glucosyltransferase and O-xylosyltransferase, occurring in seeds of lima bean (Phaseolus lunatus) and bean (Phaseolus vulgaris), respectively. Recently, the ZOG1 gene (zeatin O-glucosyltansferase) was isolated from P. lunatis (). Based on the ZOG1 sequence, the ZOX1 gene (zeatin O-xylosyltransferase) was cloned from P. vulgaris. ZOX1 contains an open reading frame of 1362 bp that codes for a 454-amino acid peptide of 51 kD. The recombinant protein has properties identical to the native enzyme: it catalyzes O-xylosylzeatin formation with UDP-Xyl as a glycosyl donor but does not recognize UDP-Glucose as a substrate. The ZOX1 and ZOG1 genes exhibit 93% identity at the nucleotide level and 90% similarity at the amino acid level. Neither gene contains introns. These zeatin-specific genes and their promoters will be useful for studies of the regulation of active versus storage forms of cytokinins. Comparison of sequences encoding similar enzymes with distinct substrate specificity may lead to identification of epitopes specific to cytokinin and glycosyl donor molecules.

Cytolocalization of zeatin O-xylosyltransferase in Phaseolus.

Zeatin O-xylosyltransferase (EC 2.4.2.-) mediates the formation of O-xylosylzeatin from trans-zeatin and UDP-xylose in immature seeds of Phaseolus vulgaris. Tissue printing with a monoclonal antibody specific for the enzyme and a cDNA probe demonstrated that the enzyme was primarily localized and synthesized in the endosperm. Immunolocalization performed on monolayer endosperm at the free-nuclei stage and on EM sections demonstrated that the enzyme was associated with the nucleus as well as with the cytoplasm. Immunoanalysis of nuclear fractions revealed that the enzyme was retained in the nuclear pellet. Western analysis also showed that the enzyme was present in the nuclei of cotyledons and endosperm callus. The findings suggest that the enzyme may be involved in the nuclear-cytoplasmic transport of cytokinins and related molecules or, possibly, with chromatin of rapidly dividing cells.

Isolation of a cytokinin gene, ZOG1, encoding zeatin O-glucosyltransferase from Phaseolus lunatus.

Zeatin is the most active and ubiquitous of the naturally occurring cytokinins. The O-glucoside of zeatin, found in all plants examined, is considered to be important in cytokinin transport, storage, and protection against cytokinin oxidases. The enzyme UDPglucose:zeatin O-glucosyltransferase (EC 2.4.1.203) was previously isolated from Phaseolus lunatus seeds. Immunoscreening of an expression library with monospecific antibody resulted in the isolation of a cDNA encoding the enzyme. The recombinant protein efficiently converts labeled zeatin to O-glucosylzeatin and has properties similar to the native enzyme. The cDNA of 1.5 kb contains an ORF encoding a 51. 4-kDa polypeptide of 459 amino acids. The sequence is unique based on a BLAST search of data bases. The genomic sequence, isolated with PCR using specific primers based on the cDNA sequence, does not contain introns. The cloning of this gene provides the tools for further study of the regulation of cytokinin metabolism and analysis of the precise role of O-glucosylzeatin in plant development.

Induction of cytokinin autonomy by N,N'-diphenylurea in tissue cultures of Phaseolus lunatus L.

The ability of N,N'-diphenylurea (Ph(2)urea) to substitute for cytokinin-active adenine derivatives in promoting callus growth of Phaseolus lunatus has been examined. In general, Ph(2)urea stimulated callus growth at high concentrations, although the growth of most callus tissues was irregular. Variability in the sensitivity and uniformity of the growth response to Ph(2)urea was found among different genotypes of P. lunatus. Most importantly, tissues cultured on Ph(2)urea-containing medium for one passage had acquired the ability to proliferate in subsequent passages in the absence of either Ph(2)urea or cytokinin-active adenine derivatives. Corresponding tissues maintained on kinetin-containing medium remained cytokinin-dependent. It appears that the effect of Ph(2)urea in promoting the growth of P. lunatus callus tissue resides in its ability to induce cytokinin autonomy. This result suggests that the cytokinin activity of Ph(2)urea may be due to promotion of endogenous cytokinin biosynthesis in the bioassay systems in which it is active.

Analyses of Phaseolus vulgaris L. and P. coccineus Lam. hybrids by RFLP: preferential transmission of P. vulgaris alleles.

Restriction fragment length polymorphism (RFLP) was determined among P. vulgaris genotypes and Phaseolus species using 19 probes. The incidence of polymorphism was high (70-86%) between species, but relatively low (22-26%) between genotypes of P. vulgaris. Suitable probes were identified for the analysis of P. vulgaris and P. coccineus hybrids. The segregation pattern in F2 populations was Mendelian for two probes (LHB and VEE20) and non-Mendelian for GS-g, CHS, and CHI. Statistical analyses indicated gametic selection with preferential transmission of the P. vulgaris alleles, which may account for the selective recovery of P. vulgaris progeny types observed earlier. The available hybrids of P. vulgaris and P. coccineus and the high degree of interspecific RFLP will facilitate the construction of a linkage map for Phaseolus.

Cytokinin metabolism in phaseolus embryos : genetic difference and the occurrence of novel zeatin metabolites.

The metabolism of trans-[8-(14)C]zeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests and GC-MS analyses. Five major metabolites were recovered from P. vulgaris embryo extracts: ribosylzeatin, ribosylzeatin 5'-monophosphate, an O-glucoside of ribosylzeatin, and two novel metabolites, designated as I and II. Based on results of degradation tests and GC-MS analyses, I and II were tentatively identified as O-ribosyl derivatives of zeatin and ribosylzeatin. In embryos of P. lunatus, however, metabolites I and II were not present. The major metabolites were ribosylzeatin, ribosylzeatin 5'-monophosphate, and the O-glucosyl derivatives of zeatin and ribosylzeatin. The zeatin metabolites recovered were the same for embryos of different sizes but their quantities varied with embryo size and incubation time. The genetic differences appear to be embryo-specific and may be useful in the studies of the possible relationship between abnormal interspecific hybrid embryo growth and hormonal derangement in Phaseolus. In addition, analyses of both organized (intact) and unorganized (callus) tissues of the same genotype may provide an opportunity to address the problem of differential expression of genes regulating cytokinin metabolism during plant development.