Esther Simón

CIRCADIAN RHYTHM OF Robinia pseudoacacia LEAFLET MOVEMENTS: ROLE OF CALCIUM AND PHYTOCHROME

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase‐response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl2 (10 mM), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase‐response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 mM), and a calcium channel blocker, LaCl3 (10–50 mM), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.

Seasonal changes in production and nutrient content ofCynodon dactylon (L.) Pers. subjected to water deficits

Seasonal changes in water relations, production and mineral composition were studied in a sward ofCynodon dactylon (L). Pers. subjected to water deficits during a dry summer, and at recovery in autumn. The experiment was carried out under Mediterranean field conditions. Water deficits during summer reduced total dry matter production by 60%, but in autumn there were no differences between treatments. Compared to well-watered sward, the sward grown under drought showed an increase in potassium, calcium and nitrogen of 55, 10 and 10% respectively. These differences decreased with the arrival of autumn rains. Leaf osmotic potential (ΨΠ) fell during the dry summer to −2.8 MPa in well-watered plants and to −4.2 MPa in stressed plants. In autumn there were no differences between treatments. Nevertheless, relative water content (RWC) only decreased to 0.86 in droughted plants. In summer potassium contributed to the osmotic adjustment. In contrast, under water deficits a decrease of 71% in sodium and, to a lesser but significant extent decreases in phosphorus, magnesium and chlorine was observed. Nitrogen, phosphorus and sulphur showed low concentrations during summer and increased in autumn.

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N2 fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N2 fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.

Nitrate Reductase Activity and Nitrate Content under Two Forms and Three Levels of Nitrogen Nutrition in Lolium perenne L.

Summary The main objectives of this work were to study the effect of different nitrogen sources on plant growth, NO - 3 -N accumulation, and on the expression of nitrate reductase activity in Lolium perenne L. leaves. Plants were grown under field conditions in a sandy loam soil (PH = 7.8) supplemented with a minus N solution (control plots) or with either NaNO 3 or (NH 4 ) 2 SO 4 applied at three levels (250, 500 or 1000 kg N/ha/year) as N sources. Nitrate reductase was assayed by an in vivo method. Although significant levels of nitrate reductase activity of NH + 4 -N and NO - 3 -N-dependent plants grown at three levels of fertilizer N were demonstrated throughout this work, the highest activities were found in plants dressed with 1000 kg NH + 4 -N/ha/year. Nitrate reductase in leaves of L. perenne showed two peaks during their growth. The first peak occurred in midsummer (July-August) and the second peak at the end of October, when rates of soil nitrification were the highest throughout the year. Higher contents of nitrates were always obtained in plants supplied with nitrate compared with those treated with ammonium. Nitrate tended to accumulate in late spring and autumn, presenting the lowest concentrations in midsummer, when growth is reduced by high temperatures. Plants grown on NH + 4 -N showed significantly greater amounts of dry matter, whereas the ones grown on NO - 3 -N accumulated more nitrates.

Effects of Calmodulin Inhibitors and Blue Light on Rhythmic Movement of Robinia pseudoacacia Leaflets

Abstract— The effect of several calmodulin (CAM) antagonists, blue light and an intracellular calcium inhibitor, on the circadian rhythm of Robinia pseudoacacia leaflet movement has been studied. The CAM antagonists, chlorpromazine (CPZ), trifluoperazine (TFP), calmidazolium and N‐(6‐aminohexyI)‐5‐chloro‐1‐naphthalenesulfonamide (W7) shifted the phase of the circadian rhythmic movement while W5, an inactive analogue of W7, had no effect. Two hour pulses of calmidazolium (10–50 μM) gave rise to a phase‐response curve with maximum advances (up to 9 h) at circadian time (CT) 6 and maximum delays (up to 7 h) at CT 22. No effect was found on transition from subjective day to subjective night and vice versa. The TFP (10–50 μM), applied as 2 h pulses during the circadian cycle, shifted the phase of the circadian leaflet movement and also produced maximum advances in the middle of subjective day. Two hour blue light pulses shifted the phase of leaflet rhythmic movement. The phase‐response curve obtained showed maximum advances (up to 5 h) in the middle of subjective day and maximum delays on transition from subjective day to subjective night. Two hour pulses of 50 μM 8‐(diethylamino)octyl 3,4,5‐trimethoxybenzoate hypochloride (TMB‐8), an intracellular calcium inhibitor, caused the same type of phase‐response curve, with maximum advances and delays occurring at the same time as those produced by blue light. These results indicate that CAM might be involved in controlling the circadian oscillator that drives Robinia leaflet movement. The relationship between CAM and calcium with red and blue light is discussed.

MAP-kinase activity in etiolated Cucumis sativus cotyledons: The effect of red and far-red light irradiation

Phytochrome (phy) signalling in plants may be transduced through protein phosphorylation. Mitogen-activated protein kinase (MAP-kinase, MAPK) activity and the effect of R (red) and FR (far-red) light irradiation on MAPK activity were studied in etiolated Cucumis sativus L. cotyledons. By in vitro protein phosphorylation and in-gel assays with myelin basic protein (MBP), a protein band (between 48 and 45 kDa) with MAPK-like activity was detected. The addition to the phosphorylation buffer of specific protein phosphatase (PTP) inhibitors (Na(3)VO(4) and NaF) and genistein, apigenin or PD98059 as MAPK inhibitors allowed us to confirm the MAPK activity of the protein band. Irradiation of etiolated cotyledons with FR light for 5, 10 or 60 min rapidly and transiently stimulated the MAPK activity of the protein band. This suggests that there was a very low fluence response (VLFR) of phys. In addition, 15 min of R light irradiation or a sequential treatment of 15 min of R plus 5 min of FR also increased MAPK activity. The stimulatory effect of R light was also attributed to the same photoreceptor, which suggests that MAPKs are involved in phytochrome signal transduction. Protein immunoprecipitation and immunoblotting analysis with the polyclonal antibody anti-pERK1/2 (Tyr 204) and the monoclonal antibody anti-phosphotyrosine PY20 allowed us to recognize the above mentioned protein band as two proteins with molecular masses (M(r)) of approximately 47 and 45 kDa, and MAPK activity. The biochemical and immunological properties showed by the proteins detected indicated that they were members of the MAPK family phosphorylated in tyrosine residues.

Phytochrome Signal Transduction in Cucumber Cotyledons

Nitrate reductase (NR) is the key enzyme in nitrate assimilation in plants. The specific calcium requirement by NR and its role in enzyme function are not well stablished. Previous experiments showed that Pfr (far-red light-absorbing form of phytochrome) promoted NRA in etiolated cotyledons of Cucumis sativus through a red-far-red reversible response, and an increase in cytoplasmatic calcium mimicked the Pfr response (Bergareche et al 1994). PI turnover is a major mechanism for transmembrane signalling in response to external stimuli (Lehle, 1990). Several lines of evidence support the presence of plant PI, the enzymes involved in their metabolism and a Ca2+-regulated amplification system responsive to IP3. It has been proposed that inositol phospholipids are hydrolyzed in the light and their products, IP3 and diacylglycerol (DAG), increase cytosolic Ca2+ and activate kinases, respectively (Berridge 1987). Furthermore, Pfr affects the levels of PI in etiolated leaves of Zea mays (Guron et al 1992). PI turnover replaces the light requirement for the induction of NR in maize (Chandok and Sopory 1994).

Microautoradiographic localisation of [3H]sucrose and [3H]mannitol in Robinia pseudoacacia pulvinar tissues during phytochrome-mediated nyctinastic closure.

Summary.We have analysed the incorporation of [3H]sucrose and [3H]mannitol in pulvinar motor cells of Robinia pseudoacacia L. during phytochrome-mediated nyctinastic closure. Pairs of leaflets, excised 2 h after the beginning of the photoperiod, were fed with 50 mM [3H]sucrose or [3H]mannitol, irradiated with red (15 min) or far-red (5 min) light and placed in the dark for 2–3 h. Label uptake was measured in whole pulvini by liquid scintillation counting. The distribution of labelling in pulvinar sections was assessed by both light and electron microautoradiography. [3H]Sucrose uptake was twice that of [3H]mannitol incorporation in both red- and far-red-irradiated pulvini. In the autoradiographs, [3H]sucrose and [3H]mannitol labelling was localised in the area from the vascular bundle to the epidermis, mainly in vacuoles, cytoplasm, and cell walls. Extensor and flexor protoplasts displayed a different distribution of [3H]sucrose after red and far-red irradiation. Far-red light drastically reduced the [3H]sucrose incorporation in extensor protoplasts and caused a slight increase in internal flexor protoplasts. After red light treatment, no differences in [3H]sucrose labelling were found between extensor and flexor protoplasts. Our results indicate a phytochrome control of sucrose distribution in cortical motor cells and seem to rule out the possibility of sucrose acting as an osmoticum.

Casein kinase activity in etiolated Cucumis sativus cotyledons

Two calcium- and light-dependent protein kinases have been reported in etiolated Cucumis sativus cotyledons (Vidal et al. 2007). In the present work, we studied casein kinase (CK) activity in etiolated cucumber cotyledons of in-gel and in vitro kinase assays, using specific CK inhibitors, and ATP and GTP as phosphate donors. Two proteins with CK activity were detected in both casein gels and autophosphorylation assays. One of them, with a molecular mass of approximately 36 kDa, showed biochemical CK1 characteristics: it was inhibited by specific CK1 inhibitors and only used ATP as phosphate donor. The second, with a molecular mass of approximately 38 kDa, had CK2 characteristics; it used both ATP and GTP as phosphate donors, was inhibited by all specific CK2 inhibitors, and was recognized by a polyclonal antibody directed against the alpha catalytic subunit of a CK2 from tobacco. The kinase activity of the CK2 detected in etiolated cucumber cotyledons showed circadian rhythmicity in both in vitro and in-gel casein phosphorylation and in autophosphorylation assays. Thus, our results suggest that the CK2 of approximately 38 kDa could be related to the circadian oscillator of C. sativus cotyledons.

Fundamental and Applied Aspects of Somatic Embryogenesis in Araujia sericifera

Araujia sericifera belongs to the Asclepiadaceae family. It has twining stems up to 10 m high, and the leaves are ovate-oblong, acurate, truncate at the base and white-tomentose beneath. Cymes are few-flowered with long peduncles. The flowers which are in axillary cymes with single corola and five segments, are white and pink striped and give off a delicate fragance. Flowers are always insect pollinated. The fruit is oblong and pruinose, containing many seeds with terminal filaments. More botanical information can be attained from Cabrera and Zardini (1978), Fiori (1925) or Flora Europea, vol.III.