Effect of Cytokinins on Photosynthetic Pigments and Chlorophyllase Activity in in Vitro Cultures of Axillary Buds of Dianthus caryophyllus L.

The effect of benzyladenine (BA) and two phenylurea cytokinins, N-phenyl-N′-(2-chloro-4-pyridyl)urea (4-PU-30) and thidiazuron (TDZ), on the growth, photosynthetic pigment content, and activity of chlorophyllase (chlorophyll-chlorophylliodhydrolase, EC 3.1.1.14) of in vitro cultures of carnations was studied. All cytokinins caused a rise in the fresh weigth and a drop in the dry weight of leaf mass produced by the explanted buds. Both 4-PU-30 and TDZ increased the chlorophyll content and this correlated with changes in chlorophyllase activity. The effect of 4-PU-30 and TDZ was similar to that caused by BA but at 10-fold or 100-fold lower concentrations. The application of higher concentrations of the phenylurea cytokinins caused an increase in the chlorophyll a/chlorophyll b ratio. However, at equimolar concentrations, the purine and both phenylurea cytokinins had opposite effects, probably indirect and related to some malformations caused by phenylureas. 4-PU-30 increased, but TDZ decreased, photosynthetic membrane stability, which argues for a different molecular organization of the chloroplast membranes. Received February 26, 1996; accepted May 30, 1997     

On the Reversible Conjugation of [17-D2]GA20 in Seedlings of Phaseolus coccineus L.

After administering [17-D₂]GA₂₀ to Phaseolus coccineus L. cv. Preisgewinner seedlings, [17-D₂]GA₂₀-O-glucoside was identified by liquid chromatography (LC)/ESI-tandem mass spectrometry (MS). Likewise, by LC/ESI-tandem MS the metabolic formation of [17-D₂]GA₂₀ glucosyl ester was established. The application of both [17-D₂]-labeled GA₂₀ 13-O-glucoside and GA₂₀ glucosyl ester to Phaseolus coccineus L. seedlings resulted in free [17-D₂]GA₂₀ by gas chromatography/MS. The results demonstrate that conjugation of GA₂₀ and the reconversion of the glucosyl conjugates are concomitant processes in plants. Received October 27, 1998; accepted August 12, 1999     

Sunfleck dynamics and canopy structure in a Phaseolus vulgaris L. canopy

 Photosynthetic photon flux density (PPFD) fluctuations were quantified in crops of beans (Phaseolus vulgaris L.) in the field as the canopy developed between July and October. Two different methods were used to select sunflecks and shadeflecks. Four ranges of zenith angles (60–70°, 50–60°, 40–50° and 30–40°) were selected for analysing PPFD fluctuations. At the base of the canopy, sunflecks contributed 18%, 53%, 10% and 4% during the 1st, 3rd, 5th and 7th week of growth, respectively. At a height of 20 cm above the soil surface, the respective contributions were 28% and 21% during the 6th and 7th weeks. Sunfleck lengths of 0–5 s were the most frequent, with the greatest number being found with smaller zenith angles. The proportion of short duration sunflecks increased as the growth period advanced. The number of long sunflecks decreased with time, with very few longer than 100 s by the 5th and 7th weeks. The distributions of sunfleck irradiance were similar to normal distributions and irradiance ranged in μmol m⁻² s⁻¹ from 600–900, 800–1500 and 1000–1600 respectively at zenith angles of 50–60°, 40–50° and 30–40°. A multiple regression showed that short sunflecks (<100 s) depended on zenith angle, plant height, and leaf and stem area index (L _s ), whereas long sunflecks (>100 s) depended on zenith angle and L _s. Shadefleck distributions were similar to those for sunflecks but there were fewer of the shortest examples and more of the longest. The best statistical distribution to describe sunflecks and shadeflecks was the gamma distribution, which could provide the basis for the future development of a good model for sunfleck and shadefleck distributions. Received: 10 November 1997 / Accepted: 2 April 1998     

Physiological and Biochemical Role of Brassinosteroids and Their Structure-Activity Relationship in the Green Alga Chlorella vulgaris Beijerinck (Chlorophyceae)

This paper studies the influence of the 7-oxalactone type of brassinosteroids (BRs) and 6-ketone upon the biological activity of the alga Chlorella vulgaris (Chlorophyceae). The results of the study indicate significant differences in the growth and metabolism of C. vulgaris cells caused by the different chemical structures of the BRs used. The most significant differences in the stimulation of the growth of the biomass and metabolites contained in it were caused by structural differences in the B ring of BRs. It was found that in C. vulgaris 7-oxalactone type of BRs [brassinolide (BL) and its derivatives] are more active than 6-ketone type of BRs [castasterone (CS) and its derivatives]. It was found that BRs used within the range of concentration of 10⁻¹² to 10⁻⁸ m stimulate two- to threefold the growth and division of C. vulgaris cells. The most stimulating influence upon the number of the algal cells and the phosphorus, chlorophyll, and monosaccharides contained in the alga, as well as the intensity of the photosynthesis, and sugar and glycolate excretion was demonstrated by BL at a concentration 10⁻⁸ m in the 36th h of cultivation. HomoCS was characterized by the lowest biological activity. In turn, after the 48th h an inhibition of the rate of growth and development of the alga takes place. In the range from 10⁻⁷ to 10⁻⁶ m the inhibition of growth and development of the alga was manifested by BRs. During the further toxic activity of BRs the cells of C. vulgaris undergo complete degradation. In turn, in concentrations lower than 10⁻¹² m, BRs do not exert any biologically significant influence upon C. vulgaris cells. On the basis of the study, the biological activity of BRs was arranged in the following order: BL > 24-epiBL > homoBL > CS > 24-epiCS > homoCS. Received July 21, 1997; accepted April 7, 1998     

Leaf orientation and distribution in a Phaseolus vulgaris L. crop and their relation to light microclimate

Changes in canopy structure parameters (leaflet orientation, leaflet inclination and leaf area index) were measured in crops of beans (Phaseolus vulgaris L.) in the field as the canopy developed between July and October. These changes were compared with the corresponding changes in seasonal light transmission. The beans showed clear heliotropic behaviour, with preferential orientation of leaflets towards the sun’s beam, especially on sunny days. Nevertheless a significant proportion of the leaves pointed in other directions, with as much as 20% oriented towards the north. The highest proportion of leaf inclinations was in the range 30–40° on cloudy days and between 40° and 50° on sunny days. Two methods were compared for assessing changes in light transmission: (a) the use of a Sunfleck Ceptometer and (b) the use of continuous records obtained with sensors installed in the canopy. Over the growth period studied, the total of the leaf plus stem area indices (L _S) increased from 0.26 to 5.2 with the transmission coefficient (τ) for photosynthetic photon flux density (PPFD), obtained using the Ceptometer, correspondingly decreasing from 0.72 to 0.05, and the canopy extinction coefficient decreasing from 1.4 to 0.62. The continuous records of light transmission gave generally similar estimates of τ. Some contrasting leaf angle distribution functions were compared for estimation of L _S from the light measurements. The best leaf angle function to predict L _S from the observed light transmission was a conical function corrected by the degree of heliotropism. Received: 27 January 1999 / Accepted: 11 June 1999     

Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998     

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.     

Actin expression in germinating seeds of Phaseolus vulgaris L.

Actin was present at very low levels in the seeds of common bean (Phaseolus vulgaris L.) compared with those from other species, and was observed mostly in the embryo. A time-course of actin expression in germinating bean seeds revealed an induced expression of both the mRNA and protein. Initially, the actin mRNA in seeds was barely detectable by northern blot analysis. However, there was a substantial increase in the expression of the actin mRNA at 24, 48 and 72 h after imbibition, compared with an internal control consisting of a late-embryogenesis-abundant (LEA) type IV gene from P. vulgaris. An increase in the amount of actin in total seed extracts that parallelled that of the mRNA was detected by western blotting starting at 24 h after imbibition. This increase was more apparent when the embryo alone was analyzed. Two-dimensional western blots initially revealed three actin isoforms with isoelectric points (pIs) of approximately 5.6, 5.7 and 5.8, the amounts of which increased within a 48-h period, when a new minor isoform of pI approximately 5.5 appeared; however, after 72 h, the pI-5.8 isoform had almost disappeared and the pI-5.5 isoform had disappeared completely, indicating that these two minor isoforms are expressed transiently. These results indicate that actin is at very low levels in the dry seed but undergoes an increased and differential expression during imbibition, an event probably required to carry out all the necessary functions for germination. Received: 21 July 1998 / Accepted: 1 September 1998     

矮生菜豆豆荚产量构成因素的通径分析

以8个矮生菜豆品种为材料,研究了单株豆荚产量与其主要构成因素的关系,结果表明,单株豆荚产量及作为豆荚产量构成因素的主茎高、分枝数、花序数、豆荚数、豆荚长和豆荚重在品种间均存在极显著的差异;但这些产量构成因素与单株豆荚产量之间,无论是表型相关系数还是遗传相关系数或是环境相关系数均未达到显著水平。通径分析结果显示,一个与单株豆荚产量关系密切的性状。其对单株豆荚产量的效应总是由于存在一个或多个负向的间接通径系数而被削弱。从而掩盖了该性状对单株豆荚产量的遗传效应,从表型和遗传通径系数看,对单株产量最为重要的是单株结荚数及单株花序数。单株分枝数、主茎高和豆荚重其次。豆荚长则较为次要。根据通径分析结果,就矮生菜豆丰产性育种中各有关性状的选择进行了讨论。     

Levels of Cytokinins in the Ovules of Cotton Mutants with Altered Fiber Development

Endogenous levels of cytokinin and abscisic acid (ABA) were determined in ovules of normal cotton (TM-1) and four fiber differentiation mutants (n2, Ligon lintless, H10, and Xu142) before and after flowering by enzyme-linked immunosorbent assays. The fluctuation patterns of ABA levels in ovules of normal cotton and mutants were similar. At the fiber elongation stage, ABA content was low, and from 1 day after flowering, the ABA content decreased steadily. On the other hand, the peaks of isopentenyladenine and isopentenyladenosine in ovules of TM-1 were observed 1 day before flowering. The level of cytokinins decreased after flowering in TM-1, whereas in the mutants it increased steadily. These results indicate that endogenous ABA is probably not the main inhibitor for fiber elongation and that endogenous cytokinins likely play a dual role in fiber development. Before flowering, cytokinins function as one of the stimuli for the initiation of fibers, but after flowering, cytokinins inhibit fiber growth. Received February 18, 1997; accepted June 11, 1997     

Analysing partitioning of recently fixed and of reserve carbon in reproductive Phaseolus vulgaris L. plants

Abstract. Partitioning of recently-fixed carbon among plant organs and subsequent distribution of reserve carbon were studied by supplying whole shoots of bean plants ( Phaseolus vulgaris L.) with ¹⁴C-labelled CO₂ of constant specific radioactivity throughout a photoperiod. The gain of tracer carbon in each part revealed net accumulation of recently-fixed carbon from direct fixation, import or both. Growth rate coefficients describing the present pattern of plant growth were calculated from ratios of tracer carbon to total carbon present in plant organs and were used to project future plant form. The period 10–20 d after the start of flowering was marked by a major increase in partitioning of recently-fixed carbon to reproductive growth. Growth rates for the plant and its parts during this period were projected on the basis of growth rate coefficients and were found to be similar to rates measured by gravimetric growth analysis. Changes in tracer carbon recovered in individual organs after chase periods of various lengths revealed net decreases for leaves and stems. About 9% of the carbon distributed to fruits came from reserves even in the absence of obvious stress. Respiratory loss during the chase period was determined from the progressive drop in recovery of the original tracer carbon. The methods are being applied to measure current net accumulation rates in studies of sink organ physiology, and to compare partitioning of recently-fixed and of stored carbon in several plant species under defined growth conditions.     

Hormonal Control of Gene Expression During Reactivation of the Cell Cycle in Tobacco Mesophyll Protoplasts

The roles of auxin and cytokinin in cell cycle reactivation were studied during the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacum. Using hormone delay and withdrawal studies we found that auxin was required by 0–4 h of culture, whereas cytokinin was not required until hour 10–12, which is 6–10 h before S phase. Cycloheximide blocks division, indicating that protein synthesis is required. In an effort to detect a molecular response to either hormone, we examined the expression of the cell cycle marker, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. However, cdc2 was partially induced by either auxin or cytokinin alone, suggesting that cdc2 expression is not the primary target of either hormone. Our hormone delay experiments suggest that there are separate signal transduction pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechanisms for these distinct pathways remain to be elucidated. Received November 4, 1997; accepted October 7, 1998     

UV-B increases the harvest index of bean (Phaseolus vulgaris L.)

The effects of small changes in natural UV-B on the photosynthesis, pigmentation, flowering and yield of bean plants (Phaseolus vulgaris L. var. Label) were studied. To obtain a relatively natural growth environment, the plants were grown in small, half-open greenhouses of UV-transmitting Plexiglas of different thickness (3 and 5 mm), resulting in an 8% difference in the weighted UV-B reaching the plants. Although the UV-B doses used did not significantly influence photosynthesis on a leaf area basis during vegetative growth, important changes in biomass allocation were noted. A UV-B-O induced reduction in leaf area during the period of vegetative growth resulted in decreased dry weight after 57 d. During the flowering and pod-filling stages (57–79.d after planting), however, plants grown at high UV-B retained their photosynthetic capacity longer: maximal photosynthesis, chlorophyll and N content of the leaves were higher under the higher UV-B dose at a plant age of 79 d. Combined with an increased allocation under the higher UV-B dose of both N and biomass to the pods, this resulted in a small increase in yield and an important increase in harvest index with increased UV-B.     

Characterization of Rhizobium etli and other Rhizobium spp. that nodulate Phaseolus vulgaris L. in an Austrian soil

Phaseolus vulgaris L. (common bean) is nodulated by rhizobia present in the fields around the Seibersdorf laboratory despite the fact that common bean has not been grown for a long time. Using PCR analysis with repetitive primers, plasmid profiles, nifH profiles, PCR-RFLP analysis of the 16S rRNA gene and of the 16S rRNA-23S rRNA intergenic spacer and the nodulation phenotype, two well-differentiating groups could be distinguished. One group showed high similarity to Rhizobium sp. R602sp, isolated from common bean in France, while the other showed the same characteristics as R. etli . We detected little variation in the symbiotic regions but found higher diversity when using approaches targeting the whole genome. Many isolates obtained in this study might have diverged from a limited number of strains, therefore the Austrian isolates showed high saprophytic and nodulation competence in that particular soil.     

QTL mapping for nodule number and common bacterial blight in Phaseolus vulgaris L.

A recently developed bean RFLP linkage map was used to identify genetic elements affecting quantitative trait loci (QTLs) in two contrasting common bean genotypes, BAT-93 and Jalo EEP558, under two levels of mineral nitrogen: low – 0.25 mM NH4NO3 and a high – 6 mM NH4NO3. QTLs affecting nodule number (NN) and response to Xanthomonas campestris bv. phaseoli, which causes common bacterial blight (CBB) were identified and mapped. Analyses of 70 F2-derived F3 families, using the F1, the two parents, and a nodulation-defective mutant (Nod-) inoculated with R. tropici UM1899 under both levels of N showed significant differences (P#60;0.0001) among the F3 families for NN.Under low N, three genomic regions influenced both traits, with seven linked markers. In three of the six regions influencing NN, higher NN was associated with the Jalo EEP-558 allele, whereas in only two regions was the BAT-93 allele associated with higher NN. One-way analysis of variance, with each marker as the independent variable and NN as the dependent variable, and interval mapping analysis identified four QTLs, which accounted for 45% of the total variation, and two additional QTLs near to yet unassigned loci. In linkage group D7, one QTL mapped to the same region as a QTL for CBB.Under high N, three additional regions were linked to NN, one where the BAT-93 allele was closely associated with CH18 (chitinase), and the others where the Jalo EEP-558 allele was associated with CHS (chalcone synthetase) and PAL-1 (phenylalanine ammonia lyase). Four regions for CBB were mapped adjacent to or in the same region as a QTL for NN. Thus, N showed dual and opposite effects on the expression of NN and CBB. Analysis of these RFLP markers revealed these hidden favorable alleles and can serve as an indirect selection tool to increase NN and resistance to CBB.     

Polyamines in Helianthus annuus L. during Germination under Salt Stress

The level of the three main polyamines putrescine, spermidine, and spermine and the biosynthetic enzyme arginine decarboxylase (ADC) decreased in Helianthus annuus L. seedlings subjected to increasing (50, 100, and 150 mm) NaCl concentrations. The pattern of polyamines in control plants increased during the initial 72 h and then reached a plateau. The putrescine level showed an increase of 370% after 72 h of development. The lower salt treatment slightly diminished the overall polyamine content. The highest NaCl concentration (150 mm) induced a strong putrescine diminution (from 381 to 78.9 nmol g⁻¹ FW) at 72 h whereas a small decrease in ADC activity was detected. ODC was detected in neither control nor treated plantlets during the experimental period. The level of spermidine also decreased, but the magnitude of the decay was less pronounced than putrescine. The fact that ODC was not detected and ADC activity followed a pattern similar to that of putrescine led us to suppose that the variation in putrescine content could be attributed entirely to the decrease in ADC activity. α-Difluoromethylarginine and α-difluoromethylornithine (ADC and ODC inhibitor, respectively) did not inhibit but delayed the onset of germination of sunflower seeds, and α-difluoromethylornithine increased the content of spermidine and spermine. The present data suggest that polyamines could be involved in the germination process of H. annuus seeds and in response to salt stress. Received April 14, 1997; accepted July 10, 1997     

Effect of phosphorus deficiency on growth angle of basal roots in Phaseolus vulgaris

Root architectural plasticity might be an important factor in the acquisition by plants of immobile nutrients such as phosphorus (P). In this study, we examined the effect of P availability on the orientation of basal roots with respect to gravity, and thereby on the growth angle of these roots of common bean (Phaseolus vulgaris L.). In one set of studies the growth angle of basal roots of bean seedlings was measured over time. Sixteen bean genotypes were examined; six showed a decrease in root orientation with respect to gravity in low P media, one increased orientation, and nine showed no difference within 5 d of basal root emergence. Bean taproots also showed decreased root orientation with respect to gravity in low P. Growth angle after 5 d was correlated with field performance of contrasting genotypes in low P tropical soils. Mineral deficiencies other than P did not cause changes in root angle. In a split pouch system that provided high or low P solution to different parts of the root system, the decrease in root angle in low P was found to be a response to global P availability, and not local to the portion of the root system in low P. Effects of P availability on root angle were associated with reduced shoot P concentration, but preceded effects on plant biomass accumulation and leaf area expansion. Results from growth pouches for genotype G 19833 were confirmed using a solid-phase buffered sand-culture system supplying P at three levels. Pea (Pisum sativum), soybean (Glycine max Williams), chickpea (Cicer arietinum), lima bean (Phaseolus lunatus), and lentil (Lens culinaris) were grown with and without P; soybean and pea also showed decreased basal root angles in low P.