Halcyon days with Bill Arnold

The circumstances that led to the discovery that plants luminesce after they are illuminated are described, as are other discoveries that would not have been possible were it not for the fortuitous association I had with my dear and most admirable friend, W.A. Arnold, to whom this special issue is dedicated.     

Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM plant Bryophyllum fedtschenkoi

Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg²⁺ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg²⁺ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg²⁺ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg²⁺ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg²⁺ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO₂ exchange. However, in contrast to the rhythm in CO₂ exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.     

Far-red enrichment and photosynthetically active radiation level influence leaf senescence in field-grown sunflower

Basal leaves frequently senesce before anthesis in high population density crops. This paper evaluates the hypothesis that quantitative and qualitative changes in the light environment associated with a high leaf area index (LAI) trigger leaf senescence in sunflower ( Helianthus annuus L.) canopies. Mean leaf duration (LD, time from achievement of maximum leaf area) of leaf 8 was significantly ( P < 0.05) reduced from 51 to 19 days as crop population density was increased from 0.47 to 4.76 plants m⁻². High compared to low plant population density was associated with earlier reduction in the photosynthetically active radiation (PAR) and red/far-red ratio (R/FR) reaching the target leaf. However the changes in R/FR preceded those in PAR. When the light environment of individual leaves of isolated plants growing under field conditions was manipulated using filters and FR-reflecting mirrors, LD was positively and linearly related with the mean daily PAR (MDR) received in the FR- (no FR enrichment) treatments. FR enrichment of light reaching the abaxial surface of the leaf significantly ( P < 0.05) reduced LD by 9 days at intermediate PAR levels with respect to FR-controls, but did not affect LD at the maximum PAR used in these experiments. However, when light reaching both leaf surfaces was enriched with FR, LD (for leaves receiving maximum PAR) was 13 days shorter than that of the FR- control. These results show that basal leaf senescence in sunflower is enhanced both by a decrease in PAR and by a decrease in R/FR.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Phytochrome control of short-day-induced bud set in black cottonwood

In trees and other woody perennial plants, short days (SDs) typically induce growth cessation, the initiation of cold acclimation, the formation of a terminal bud and bud dormancy. Phytochrome control of SD-induced bud set was investigated in two northern clones of black cottonwood (Populus trichocarpa Torr. & Gray) by using night breaks with red light (R) and far-red light (FR). For both clones (BC-1 and BC-2), SD-induced bud set was prevented when R night breaks as short as 2 min were given in the middle of the night. When night breaks with 2 min of R were immediately followed by 2 min of FR, substantial reversibility of bud set was observed for BC-1 but not for BC-2. By comparing the effects of the R night breaks on bud set and the length of specific internodes, we determined that the R night breaks influenced internode elongation in two opposing ways. First, the addition of a R night break to the SD treatment prevented the cessation of internode elongation that is associated with bud set. Those internodes that would not have elongated under SDs (and would have been found within the terminal bud) elongated in the R treatment. Second, the R night breaks decreased internode length relative to the long-day (LD) control. In contrast to the clonal differences in reversibility that we observed for bud set, the decrease in internode length (i.e. the second effect of R) was R/FR reversible in both clones. Based on these results, we conclude that internode elongation is influenced by two distinct types of phytochrome-mediated response. The first response is a typical response to photpperiod, whereas the second response is a typical “end-of-day” response to light quality. Our results demonstrate that SD-induced bud set in black cottonwood is controlled by phytochrome but that clonal differences have an important influence on the R/FR reversibility of this response. The availability of an experimental system in which SD-induced bud set is R/FR reversible will be valuable for studying the physiological genetics of photoperiodism in trees.     

Development of the definitive kidney in the cynomolgus monkey (Macaca fascicularis)

Abstract: Formation of the definitive kidney in Macaca fascicularis embryos was investigated using light and electron microscopy. Appearance of the definitive kidney at stage 14 was indicated by the ureteric bud invading the metanephrogenic blastema. Glomerular capillaries originate from the connective tissue that surrounds the developing renal vesicle. At 46–100 days gestational age the more developed glomeruli show thinning of the capillary endothelium, thickening of the basal membrane, and presence of pedicels, suggesting a capability of renal function.     

Effects of light, temperature, gibberellin (GA3) and their interaction on coleoptile and leaf elongation of tall, semi-dwarf and dwarf wheat

Near-isogenic wheat lines differing in height-reducing (Rht) alleles, in each of two cultivars, were used to investigate the effects of light intensity and of their interaction with temperature and GA₃ application, on the elongation of the coleoptile and the first seedling leaf. Darkness caused a conspicuous increase in the lengths of the coleoptile and of the sheath and lamina of the first leaf, in GA₃ treated and untreated seedlings of all genotypes grown at 11 and 25°C. The genotype effects and the effects of light intensity and GA₃ application on leaf length were ascribed entirely to their effects on the rate of leaf elongation since the duration of leaf elongation was not affected by these factors. Temperature elevation from 11 to 25°C caused a 55% shortening of the duration of leaf elongation and a concomitant increase in elongation rate, which diminished with increased genotypic dwarfness. Accordingly, temperature elevation resulted in a significant reduction in leaf-length of the light-grown dwarf genotypes and the dark-grown dwarf and semi-dwarf genotypes. It is suggested that this temperature × light × genotype interaction effect is due to environmental dependent upper limits of elongation rate set by the Rht alleles.Abbreviations PAR Photosynthetic Active Radiation     

The crystal violet nuclei staining technique leads to anomalous results in monitoring mammalian cell cultures

Nuclear counts determined by crystal violet staining from samples of stationary or microcarrier cultures of hybridomas, CHO or Vero cells were consistently and significantly higher than cell concentrations determined by the trypan blue or Coulter counter methods. This difference was attributed to the presence of a significant proportion of binucleated cells, which are assumed to be 35% of the cell population in the stationary phase of Vero cultures. The proportion of such cells during exponential growth was variable. However, continuous sub-culture of these cells induced a degree of synchrony during growth which resulted in a cyclic variation of the difference between the cell and nuclei counting techniques. This data indicates that care should be taken in interpreting cell culture profiles based solely on crystal violet nuclei staining counts.     

Leaf litterfall,leaf area index,and radiation transmittance in cypress wetlands and slash pine plantations in north-central Florida

The seasonal dynamics of leaf litterfall and leaf area index (LAI, all-sided basis), light penetration and the vertical distribution of surface area index, and the feasibility of estimating LAI from radiation transmittance were studied from April 1993 to March 1994 in the canopies of three cypress (Taxodium ascendens) wetlands and their surrounding slash pine (Pinus elliottii) uplands in Florida flatwoods. Annual leaf litterfall ranged from 324 to 359 g m^–2 in the wetlands, which was very close to the average for 11 sites throughout Florida of 340±26 g m^–2. The seasonal pattern of the normalized LAI obtained for the dominant tree species in the ecosystems could be used to construct the seasonal dynamics of LAI at the ecosystem scale. The vertical distribution of surface area index in the wetlands was significantly different from that in the surrounding pine uplands. The maximum LAI of cypress wetlands in this area was about 8 m² m^–2, which was higher than the maximum of slash pine plantations of 6 m² m^–2. Cypress leaves were strongly erectophile in space. Results showed that the LAI-2000 canopy analyzer could generally be used to estimate forest LAI, whether the forest canopy was closed or not, if an overall clumping index of 2.00 was applied. However, as LAI decreased, the relative error contained in the radiation-based LAI estimates increased. This indicated that foliage clumping at the stand scale was more important than that at the tree or branch scale.