Chloramphenicol effects on adventitious root production by radish hypocotyls

Abstract

The excision of the root accelerates greatly the formation of adventitious roots in the hypocotyl of etiolated radish seedlings, but if the seedlings develop in CAP 1×10^?4M, no adventitious root are induced after cutting. IAA either alone or associated with CAP, significantly increases the number of primordia in normal hypocotyls if given at the moment of cutting, while it has not stimulatory effect on the hypocotyls of seedlings grown in CAP. IAA has significant effect on both elongation and tickening of hypocotyl segments prepared from seedlings grown in CAP, and this could indicate a specific action of the inhibitor either on a particular process or on particular cells.

The endodermis and the pericycle, which are the two cell layers implicated in the formation of the adventitious roots, could be the mediators of this particular effect of CAP in rooting.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

Mode of action of natural growth inhibitors in radish hypocotyl elongation – influence of raphanusanin on auxin-mediated microtubule orientation

Raphanusanin is a plant growth-inhibiting substance which plays an important role in light growth inhibition and phototropism of radish hypocotyls. We investigated the effect of raphanusanin on indole-3-acetic acid (IAA)-mediated orientation of microtubules (MT) in the outer epidermal cells of radish hypocotyl segments using immunofluorescence microscopy. IAA-mediated MT reorientation preceded cell elongation induced by IAA. A change of IAA-mediated MT orientation from longitudinal to transverse started within less than 15 min after IAA treatment, while significant growth promotion induced by IAA was found within about 30 min. The IAA-mediated transverse MT orientations were significantly inhibited by simultaneously added raphanusanin. We also investigated the effect of raphanusanin on the MT orientation of the segments pretreated with IAA. The change of MT orientation induced by raphanusanin preceded growth inhibition of the segments. Within about 60 min after its application, raphanusanin initiated inhibition of the steady-state elongation pre-induced by IAA, while IAA-mediated transverse MT orientations started to change into longitudinal orientations within less than 30 min after application of raphanusanin. Based on these results, it is suggested that raphanusanin induces growth inhibition through interference with the auxin-mediated MT orientations.     

Mortality and growth responses of Daphnia carinata to increases in temperature and salinity

1.  Daphnia carinata King, a freshwater cladoceran, occurs in Lake Waihola, a tidal lake with seasonal fluctuations in temperature (4–21 °C), and salinity (30–2000 mg L^–1 Cl). We hypothesise that these fluctuations influence the seasonal changes in D. carinata abundance.
2. To test this hypothesis, adults and juveniles were exposed to combinations of temperature and salinity. We measured mortality of adults and juveniles, growth of juveniles over 20 days, and their age at first reproduction.
3. The salinity tolerance of adult D. carinata was temperature-dependent. Juveniles were more sensitive to salinity, but more tolerant of temperature increases.
4. Growth rates were higher at elevated temperatures, but reduced by elevated salinities. The onset of reproduction was earlier at elevated temperatures.
5. We conclude that seasonal changes in temperature and salinity contribute to seasonal population changes of D. carinata .     

Changes in trace metal species and other components of the rhizosphere during growth of radish

Changes in the properties of soil solution in the rhizosphere of developing radish plants were investigated. Variations in these properties were expected to affect the distribution and speciation of metals in the soil and soil solution. Applications of essential nutrients were linked to plant transpiration rates and prevented excess addition of nutrient ions, so that subtle changes in soil solution composition would not be obscured. Soil solution pH, the concentration of dissolved organic carbon (DOC) and the concentrations of major and trace elements in solution were found to vary over time. Strict control of fertilizer additions led to the maintenance of a relatively low ionic strength in the soil solution, and under such conditions trace metal solubility appeared to be highy influenced by the concentration of DOC. A chemical speciation analysis was performed which showed that, while dissolved Cd and Zn were largely uncomplexed in unplanted soil, Cd and Zn in the rhizosphere existed mainly as complexed forms. It is hypothesized that this is partly a result of Ca-metal-ligand equilibrium in solution, with higher Ca concentrations in unplanted soil leading to more of the Cd and Zn in solution existing in the uncomplexed state. Changes in the concentrations of uncomplexed Cd and Zn with time gave the best correlations with changes in plant uptake of these metals over time, supporting the hypothesis that plants mainly absorb the free metal ion from soil solution.     

Thiocyanate content in relation to the quality features of radish Raphanus sativus L

Relationships between radish thiocyanate content and its dry weight, the content of sugar, protein, fibre, ascorbic acid, some minerals, the incidence of plant shooting, the firmness and pithiness of storage-roots, and the ratio of leaves to storageroot (wt/wt) were investigated. The analysis of linear correlation was based on numerous data from the 4-year field experiment with six radish cultivars and different sowing and harvest dates. The content of thiocyanate in radish roots was found to be positively correlated with their dry weight, and the content of total protein, crude fibre, and soluble sugar. A strong relationship was found between the content of thiocyanate and dry weight of radish leaves. The negative correlation between the thiocyanate content in the leaves and the firmness of storageroots and the positive correlation with their pithiness might indicate the translocation of this compounds into green plant parts during the ageing of root tissue. The root thiocyanate content and the percentage of shooting correlated significantly only in the case of Tokinashi. The closeness of relations between the ratio of leaves/storage-root and thiocyanate content, though in general small, was affected also by a cultivar. A similar effect was observed for the correlations between the thiocyanate contents in leaves and storage-roots.     

Changes in thiocyanate content in some radish Raphanus sativus L. cultivars during hypocotyl-root growth

Changes in the thiocyanate content in hypocotyl-roots and leaves of radish were observed in a two-year field experiment. Six cultivars were tested: early radish (Rex and Ostergruss Różowa), Japanese radish (Tokinashi and Minowase Summer Cross F₁), and winter radish (Monachijska Biała and Murzynka). A significant diversification in thiocyanate content among cultivars, plant parts, harvest dates and observation years was found. Early cultivars contained the least amount of these compounds, Murzynka — the greatest. The content of thiocyanates in leaves was 3 – 5 times higher than that in hypocotyl-roots. The changes in the thiocyanate content during root growth showed a constant rising tendency in the case of the leaves of all cultivars and the storage organs of Murzynka.     

Response of seedlings of radish (Raphanus sativus) to osmotic shock and external hydrostatic pressure

The effects of an osmotic shock (300 m M mannitol, –0. 67 MPa) or/and increased external hydrostatic pressure on seedlings (42-h-germinated seeds) of radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) were investigated. The osmotic shock did not inhibit H⁺ extrusion and net K^- uptake, and did not affect the levels of malic acid, reducing sugars, sucrose or amino acids or of the energy charge (i. e. the synthesis of energy-rich phosphate bonds), but inhibited the synthesis of proteins. RNA and DNA, measured as incorporation of labelled precursors. When the osmotic shock was applied together with an increased external hydrostatic pressure of the same magnitude (+0. 67 MPa), the same metabolic parameters and the inhibition of synthesis of RNA and DNA were not substantially affected, while the inhibition of protein synthesis was practically reversed and the energy charge decreased; the recovery of protein synthesis was not due to a change in labelled leucine uptake capability. Increased external hydrostatic pressure alone decreased the energy charge without affecting the other parameters considered.
The possibility that protein synthesis activity is directly controlled by cell turgor pressure (internal hydrostatic pressure) is discussed.     

Increase in calmodulin level in the early phases of radish seed (Raphanus sativus) germination

Abstract Calmodulin (Cam), the heat-stable, ubiquitous, Ca²⁺-dependent regulator protein, has been purified to apparent homogeneity from germinating radish seeds (Raphanus sativus). The characteristics of radish Cam-molecular weight, absorption spectrum, Ca²⁺-dependent activation of brain phosphodiesterase (PDE)-are very similar to those described for Cam from other plant materials. Radish Cam, like other plant Cam, shows some differences to Cam of calf brain. The total amount of Cam in radish embryos at 24 h of germination is ca. 37 μg g⁻¹ fresh weight. Approximately 95% of the total amount of Cam is present in the soluble fraction (supernatant at 100,000 g). The level in the embryo axis strongly increases in the first 24 h of germination (+540%); this increase is strongly reduced when the germination is inhibited by abscisic acid (ABA). In the presence of Ca²⁺, no ‘free’ Cam (i.e. not bound to other structures) is present in the soluble fraction, suggesting that, during early germination, Cam level is a limiting factor for the activities of Ca²⁺ -Cam-dependent systems. These studies suggest that Cam plays an important role in the early phases of seed germination. An inhibitor of the Ca²⁺-Cam-dependent phosphodiesterase is present in the soluble fraction from radish embryos; this substance decreases during germination. A possible role of this inhibitor during the early germination phases is hypothesized.     

Calmodulin levels in radish (Raphanus sativus L.) seeds germinating at low calcium availability induced by EGTA treatments

Incubation of radish (Raphanus sativus L.) seeds in the presence of 1 or Smol m^?3 Ca-EGTA, which increased Ca²⁺ activity in the incubation medium (c. 0.24 or 0.37 mol m^?3 at 24 h with respect to c. 0.13 mol m^?3 in the control), did not affect germination, the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m^?3 Na-EGTA, which reduced Ca²⁺ activity in the incubation medium (20 mmol m^?3 at 24 h), decreased the total Ca²⁺ level in embryo axes (-21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m^?3 Na-EGTA (Ca²⁺ activity in the incubation medium was 0.6 mmol m^?3), the decrease in the total Ca²⁺ level was greater (c. -27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca²⁺ availability does not affect germination and suggest that the effect of Na-EGTA, at least up to 5 mol m^?3, is a result of an induction of Ca²⁺ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5-fold and 7-fold, respectively). This increase was very similar in the Ca-EGTA-treated seeds but was inhibited (c. -38%) by 1 mol m^?3 Na-EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m^?3 Na-EGTA (c. -30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci & N. Negrini (1988) Plant Physiology 88, 910–914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca²⁺ availability and that the CaM inhibitor has a role in controlling the amount of Ca-CaM available for the Ca-CaM-dependent enzymes.     

Fitness effects of mutation accumulation in a natural outbred population of wild radish (Raphanus raphanistrum): comparison of field and greenhouse environments

Spontaneous deleterious mutation has been measured in a handful of organisms, always under laboratory conditions and usually employing inbred species or genotypes. We report the results of a mutation accumulation experiment with an outbred annual plant, Raphanus raphanistrum, with lifetime fitness measured in both the field and the greenhouse. This is the first study to report the effects of spontaneous mutation measured under field conditions. Two large replicate populations (N(e) approximately 600) were maintained with random mating in the greenhouse under relaxed selection for nine generations before the field assay was performed and ten generations before the greenhouse assay. Each generation, every individual was mated twice, once as a pollen donor and once as a pollen recipient, and a single seed from each plant was chosen randomly to create the next generation. The ancestral population was maintained as seeds at 4 degrees C. Declines in lifetime fitness were observed in both the field (1.7% per generation; P= 0.27) and the greenhouse (0.6% per generation; P= 0.07). Significant increases in additive genetic variance for fitness were found for stems per day, flowers per stem, fruits per flower and seeds per fruit in the field as well as for fruits per flower in the greenhouse. Lack of significance of the fitness decline may be due to the short period of mutation accumulation, the use of outbred populations, or both. The percent declines in fitness are at the high end of the range observed in other mutation accumulation experiments and give some support to the idea that mutational effects may be magnified under harsher field conditions. Thus, measurement of mutational parameters under laboratory conditions may underestimate the effects of mutations in natural populations.     

Photosynthetic responses to salinity in two clones of Agrostis stolonifera

Abstract The response of photosynthesis, F_e, to salinity was examined in two clones of Agrostis stolonifera, one derived from a salt marsh (SM) and the other from an inland site (IL). The response of young and old leaves was examined over one week of salt treatment and changes in leaf water relations and concentrations of Na⁺, Cl^? and K⁺ ions were also monitored. Sodium⁺ and chloride^? concentrations rose much more sharply in the older leaves of both clones and F_e declined to ca. zero by Day 4. F_e in the young leaves of the SM clone was unaffected by salt whilst the young leaves of the IL clone showed a 20% reduction in F_e within 24 h of salt application. This decline was due to a decline in stomatal conductance and a reduction in the photosynthetic capacity of the mesophyll and was associated with a greater accumulation of Na⁺ and Cl^? ions and loss of K⁺ ions than in the SM clone. The significance of the salt exclusion from the young leaves of the SM clone to its growth on the salt marsh is discussed.     

Effects of pollen load size and composition on pollen donor performance in wild radish, Raphanus sativus (Brassicaceae)

A critical concern in the debate over the importance of sexual selection in plants is whether the nonrandom mating demonstrable in greenhouse crosses can occur in the field. Field populations likely experience smaller and more variable pollen load sizes than those that have been used in many greenhouse experiments. Therefore, we performed a greenhouse experiment in which we varied both pollen load size and composition in wild radish, Raphanus sativus, and examined the paternity of seeds. We used five maternal plants and four pairs of pollen donors. We were able to produce pollen loads of 40, 118, and 258 grains per stigma. The smallest of the pollen loads was scant enough to result in a slight, but significant reduction in seed number per fruit. While variation in pollen load composition significantly affected the proportions of seeds fathered by different donors, variation in pollen load size did not. The relative performance of different donors was constant across pollen load sizes, suggesting that, for this species, differential performance of pollen donors can occur at pollen load sizes that are likely to occur in field populations.     

Identification and characterization of microsatellite markers derived from expressed sequence tags (ESTs) of radish (Raphanus satvius L.)

Radish, Raphanus satvius, is a very important commercial crop in the world, especially in East Asian countries. In this study, radish microsatellite markers have been developed for the first time from expressed sequence tags (ESTs). A total of 8 primer pairs were able to produce clear amplifications, respectively, which gave 2–5 polymorphic loci between 43 cultivars of Japanese radish. Observed and expected heterozygosity ranged from 0 to 0.4186, and from 0.1779 to 0.6205, respectively. Polymorphic information content ranged from 0.171 to 0.575. The eight simple sequence repeats were also polymorphic when tested by wild radish (Raphanus raphanistrum). Deviation from Hardy–Weinberg equilibrium was analysed in three populations, and no linkage disequilibrium was observed. It is anticipated that these newly developed microsatellite markers can advance further genomic analyses. This accomplishment may in turn accelerate and simplify radish breeding programs.     

Relative growth rate correlates negatively with pathogen resistance in radish: the role of plant chemistry

Plant growth rate has frequently been associated with herbivore defence: a large investment in quantitative defence compounds occurs at the expense of growth. We tested whether such a relationship also holds for growth rate and pathogen resistance. For 15 radish (Raphanus sativus L.) cultivars, we determined the potential growth rate and the resistance to fungal wilt disease caused by Fusarium oxysporum. We subsequently aimed to explain a putative negative relationship between growth rate and resistance based on plant chemical composition. Both growth rate and resistance level varied greatly among cultivars. Moreover, there was a strong negative correlation between growth rate and resistance, i.e. there are costs associated with a high resistance level. Roots of slow-growing, resistant cultivars have a higher biomass density. Using pyrolysis mass spectrometry. we part1y explained variation in both growth rate and resistance in terms of the same change in chemical composition. Leaves of slow-growing, resistant cultivars contained more cell wall material. Surprisingly, roots of slow-growing, highly resistant cultivars contained significantly less cell wall material, and more cytoplasmic elements (proteins). We speculate that this higher protein concentration is related to high construction and turn-over costs and high metabolic activity. The latter in turn is thought to be responsible for a rapid and adequate resistance reaction, in which phenols may be involved.