Molecular cloning of Phaseolus vulgaris cDNA encoding proliferating cell nuclear antigen

A cDNA fragment encoding a common bean (Phaseolus vulgaris) proliferating cell nuclear antigen (PCNA) was isolated using rapid amplification of cDNA 3' end (3' RACE) method, cloned and sequenced. The nucleotide sequence of this clone contains an open reading frame of 798 nucleotides encoding a protein of 265 amino acids. Alignment of the common bean PCNA predicted sequence shows its high degree of identity with PCNA from other plant species. Analysis of PCNA content in the germinating embryos of common bean showed a decrease in the protein level after 60h of germination. Moreover, PCNA was not detected in the tested plant organs (root, stem, leaf and flower). The presence of PCNA in the germinating seeds and its absence from mature plants suggests that this protein plays a crucial role during early stages of plant development.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.)

A cDNA showing high sequence similarity (>70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5-untranslated region and a 317 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M _r of 35552. Alternatively an ATG situated to the 5 end of the putative start site would increase the protein size by 6 amino acids.The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.Abbreviations PAL phenylalanine ammonia-lyase - PP1 protein phosphatase 1 - Pvpp1 Phaseolus vulgaris protein phosphatase 1     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137     

DNA content of Phaseolus coccineus x P. vulgaris suspensors

Summary At the globular stage of embryo development, the level of DNA, as determined from microspectrophotometric analysis of Feulgen-stained squashes, was significantly higher in the interspecific hybrid suspensor than in suspensors from self-pollination of Phaseolus coccineus, the maternal parent. However, at the early-heart and early-cotyledonary stages of development, DNA content of interspecific hybrid suspensors was significantly lower than that of suspensors formed after self-pollination of either P. coccineus or P. vulgaris. The relationship between DNA content and suspensor cell length for P. coccineus and P. vulgaris at all developmental stages and between DNA content and cell area for P. coccineus at the early-cotyledonary stage was altered in hybrid suspensor cells. Nuclei in large cells of interspecific suspensors exhibited uneven distribution of polytene chromosomes and no clear nuclear outline.Paper number 18,470 of the Scientific Journal Series, University of Minnsota Agricultural Experiment Station, St. Paul, MN. This research was funded by the USDA CRGO under grant number USDA-85-CRCR-1-1676     

Auxin-induced assimilate translocation in the bean stem (Phaseolus vulgaris L.)

Decapitation of the fully-elongated fourth internode of Phaseolus vulgaris plants resulted in the disappearance from the internode of soluble acid invertase (EC 3.2.1.26). This loss was prevented by local applications to the internode of indol-3yl-acetic acid (IAA) and, at the point of IAA application, the specific activity of the enzyme increased by up to 3 times its initial value within 48 h of treatment. IAA applications stimulated the acropetal translocation to the internode of ¹⁴C-sucrose applied to the subtending (second) trifoliate leaf 30 h after decapitation and the start of the auxin treatment. Labelled assimilates accumulated in the IAA-treated region of the internode. Following decapitation the concentration of hexose sugars in the internode fell and that of sucrose rose substantially, but these trends were reversed by IAA treatment. However, small local accumulations of sucrose occurred at the point of auxin application where tissue concentrations of IAA were greatest (determined using [1-¹⁴C] IAA).Considerable quantities of starch were present in the ground parenchyma of the internodes at the start of the experiment but, in the absence of IAA, this was remobilised within 48 h of decapitation. IAA prevented starch loss at and below its point of application to the internode, but not from more distal tissues. Cambial proliferation, radial growth and lignification were stimulated in and below IAA-treated regions of the internode. These observations are discussed in relation to the hormonal regulation of assimilate translocation in the phloem.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Genetic control of chalcone isomerase activity in flowers of Dianthus caryophyllus

In flowers of Dianthus caryophyllus (carnation), the gene I is concerned with a discrete step in flavonoid biosynthesis, Genotypes with recessive (ii) alleles produce yellow flowers, which contain the chalcone isosalipurposide (naringenin-chalcone-2-glucoside) as the major petal pigment, but in genotypes with wild-type alleles flavonols and anthocyanins can be formed and the flowers are white or red. Enzymatic measurements on petal extracts of four strains with different flower coloration revealed a clear correlation between accumulation of chalcone in recessive genotypes and deficiency of chalcone isomerase (E.C. 5.5.1.6) activity. From the chemogenetic and enzymological evidence it can be concluded that naringenin-chalcone is the first product of the synthesis of the flavonoid skeleton and that only the conversion of naringenin-chalcone to naringenin furnishes the substrate for the further reactions to flavonol and anthocyanin.These investigations were supported by a grant from Deutsche Forschungsgemeinschaft.     

First bacterial chalcone isomerase isolated from Eubacterium ramulus

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.     

Alternaria brasiliensis sp. nov., a leaf pathogen on Phaseolus vulgaris

Alternaria brasiliensis sp. nov., was isolated from leaves of common bean, Phaseolus vulgaris L., showing punctiform non concentric leaf spots of brown color. Besides the symptomatological differences, the new Alternaria species presents distinct type of arrangement of conidia chain, body and beak size. The disease was observed in Montanha county, State of Espirito Santo, Brazil, but not yet reported in the literature. The causal agent of the disease is now described by the first time. The epithet used here is referring to the country where the species has been found. This revised version was published online in June 2006 with corrections to the Cover Date.     

Zinc mediated effects on leaf CO2 diffusion conductances and net photosynthesis in Phaseolus vulgaris L.

Supra-optimal levels of zinc in primary leaves of Phaseolus vulgaris increased the CO₂ compensation point and inhibited net photosynthesis. Leaf morphology was modified: mesophyll intercellular area, stomatal slit length and interstomatal distance were reduced, but stomatal density increased. Internal and stomatal conductances to CO₂ diffusion decreased. These changes are discussed in relation to the observed effects on leaf gas exchange and to the previously reported inhibition of different photosynthetic and photorespiratory enzymes.     

Fertile,intermediate hybrids between Phaseolus vulgaris and P. acutifolius from congruity backcrossing

Summary Recurrent or introgressive backcrossing of Phaseolus vulgaris — P. acutifolius hybrids with either P. vulgaris or P. acutifolius increased fertility, as measured by pollen stainability and seed per pod from non-manipulated flowers, while invariably resulting in loss of traits from the non-recurrent parent. When hybrids were backcrossed with each of the parent species in alternate generations (congruity backcrossing), fertility decreased in early generations but gradually increased in later generations. By the fourth or fifth generation, congruity-backcross hybrids produced numbers of seeds per pod from non-manipulated flowers comparable to those of parent species, although the percent of stainable pollen tended to be lower. Congruity-backcross hybrids were intermediate to parent species when pedigrees contained equal representation of parents but favored the majority parent when pedigrees were unbalanced. Individuals exhibiting symptoms of developmental incongruity, such as abnormal leaflet number, foliar variagation, or unusual growth patterns, occurred in each generation. However, completely new characteristics appeared after the second backcross generation, presumably due to recombination. Backcrossing to both parent species in alternate generation reversed incongruity, allowing selection for fertility without loss of traits from one of the parent species.Scientific Journal Series Paper Number 15, 671 of the Minnesota Agricultural Experimental Station     

Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity

The effect of Ca²⁺ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca²⁺ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca²⁺. Potassium and Ca²⁺ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca²⁺ in the medium. The opposite was true for Na⁺. Leakage of NO₃ ^- and H₂PO₄ ^- was increased by salinity and reduced by high Ca²⁺ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions.     

Bean lectins IV: genetic variation in the non-denatured structure of lectins from different Phaseolus vulgaris L. cultivars

Summary Variation in the native conformation of bean lectins was examined using electrophoresis of non-denatured total protein extracts and purified albumin and globulin lectin. The observed variation was related to the genetic variation reported previously for lectin polypeptide composition as revealed by two-dimensional isoelectricfocusing-sodium dodecyl sulfate polyacrylamide gel electrophoresis (IEF-SDS/PAGE). When eleven cultivars with different IEF-SDS/PAGE lectin polypeptide compositions were compared, eight had unique non-denatured lectin patterns and three had identical patterns. For some cultivars differences in non-denatured lectin patterns were observed between the purified albumin and globulin lectin preparations.     

Cis elements and potential trans-acting factors for the developmental regulation of the Phaseolus vulgaris CHS15 promoter

A nuclear factor (SBF-1) has previously been identified in Phaseolus vulgaris L. (bean) suspension cell nuclear extracts that binds in vitro to three DNase I-footprinted elements (SBF-1 boxes I, II, and III, 5 to 3) in the 5 region of the bean CHS15 (chalcone synthase) gene promoter. To define the functional role of the three SBF-1 boxes in development, we examined transgenic tobacco plants carrying a series of nested CHS15 promoter--glucuronidase (GUS) fusions for GUS activity by histochemical staining. We show that the CHS15 promoter deleted to position-173 and lacking all three SBF-1 boxes directs the same qualitative pattern of expression in initiating lateral roots and in developing seeds as the full length promoter (-326). Thus, activation of expression in these organs is mediated by sequence elements located downstream of the three SBF-1 boxes. However, specific deletions within the-326 to-173 region modulate expression. Thus, deletion of box II abolishes GUS activity in initiating lateral roots. Further deletion of box III fails to restore expression but subsequent deletion of an additional 43 bp to position-173 re-establishes expression. We show that sequence-specific DNA-binding activities consistent with these results are present in nuclear extracts of bean roots and seeds. These studies reveal cis elements within the CHS15 promoter, and potential trans factors, that permit organ- and tissue-specific developmental patterns of regulation to be combined with a flexible response to environmental cues.