Calcium changes in Robinia pseudoacacia pulvinar motor cells during nyctinastic closure mediated by phytochromes.

Potassium pyroantimonate precipitation, transmission electron microscopy, and X-ray microanalysis were used to investigate the subcellular localization of loosely bound calcium in Robinia pseudoacacia pulvinar motor cells during phytochrome-mediated nyctinastic closure. Calcium localization was carried out in pulvini collected in white light 2 h after the beginning of the photoperiod, immediately after a red light or a far-red light pulse applied 2 h after the beginning of the photoperiod and after 15 or 25 min of darkness respectively. Calcium antimonate precipitates were found in all the pulvinar tissues from the epidermis to the vascular bundle, independent of the light treatment. At subcellular level, precipitates were found mainly in the intercellular spaces, the inner surface of the plasma membrane, cytoplasm, colloidal vacuoles, and nuclei. Red light enhanced the nyctinastic closure of leaflets and caused an asymmetric distribution of cytosolic calcium precipitates between the extensor and flexor motor cells. Both the number and area of the cytosolic calcium precipitates drastically increased in the extensor cells compared to the flexor motor cells. Red light had a rapid and transient effect on the distribution of cytosolic calcium precipitates, which occurred during or at the end of the irradiation, before leaflet closure. By contrast, the distribution of cytosolic loosely bound calcium was similar between the extensor and flexor motor cells after irradiation with far-red light. Our results demonstrate that red light causes specific calcium mobilization in pulvinar motor cells and suggest the involvement of cytoplasmic Ca2+ as a second messenger for phytochrome during nyctinastic closure.

Nitric-oxide inhibits nyctinastic closure through cGMP in Albizia lophantha leaflets.

Nitric oxide (NO) is a highly reactive radical that acts as a direct or indirect cellular signalling molecule in plant growth, development and environmental responses. Here we studied the contribution of NO to the control of leaflet movements during nyctinastic closure. For this purpose, we tested the effect of NO donors and an NO scavenger, all supplied in light, on Albizia lophantha leaflet closure after transferral to darkness. Exogenous NO, applied as four donors [sodium nitroprusside (SNP), diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (DEA-NONOate), S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GS-NO)], inhibited nyctinastic leaflet closure while the application of an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] plus SNP cancelled the effect of the latter. The inclusion of Nω-nitro-l-arginine methyl ester (l-NAME) or sodium tungstate in the incubation media enhanced nyctinastic closure and also resulted in a decrease in the nitrate plus nitrite released by leaflets into the incubation solution. These results support the notion that NO is involved in regulating the nyctinastic closure of A. lophantha leaflets. Cellular perception of NO did not appear to be mediated by calcium. Pharmacological application of inhibitors of soluble guanylate cyclase (sGC) [1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ) and 6-anilino-5,8-quinolinequinone (Ly83583)], phosphodiesterase type 5 (PDE5) (Sildenafil) and the cyclic guanosine monophosphate (cGMP) analogue 8-bromoguanosine-3',5'-cyclomonophosphate sodium salt (8-Br-cGMP) indicated that cGMP was downstream of the NO signalling cascade during nyctinastic closure.

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining TILLING mutant populations.

BACKGROUND: TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. RESULTS: Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. CONCLUSIONS: This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Protein kinase activity in Cucumis sativus cotyledons: effect of calcium and light.

Light signals received by phytochromes in plants may be transduced through protein phosphorylation. Ca(2+) as second messenger was involved in phytochrome-mediated cellular events. Our experiments with Cucumis sativus cotyledons, treated with red (R) and far-red (FR) light, showed a stimulatory effect on in vitro protein phosphorylation of histone, added as exogenous substrate to the cotyledon extracts, and also modified the phosphorylation of endogenous polypeptides. The effect of light treatments was mimicked by the addition of Ca(2+) to the phosphorylation buffer, indicating phytochrome- and Ca(2+)-dependence on activity of some protein kinases (PKs). In-gel kinase assays were performed to characterize the PKs involved at the cotyledon stage of cucumber plants. Three proteins of about 75, 57 and 47kDa with PK activity were detected between M(r) markers of 94 and 45kDa. All three were able to phosphorylate histone and undergo autophosphorylation. However, only the 75 and 57kDa proteins autophosphorylated and phosphorylated the substrate in a Ca(2+)-dependent manner, and were inhibited when calmodulin (CaM) antagonists were added to the incubation buffer. Western-blot analysis with polyclonal antibodies directed against calcium-dependent protein kinase of rice (OsCDPK11) or Arabidopsis (AtCPK2) recognised 57 and 75kDa polypeptides, respectively. These results indicate the presence in cucumber cotyledons of at least two proteins (ca. 75 and 57kDa) with activity of PKs that could be calcium-dependent protein kinases (CDPKs). Both CDPKs could be modulated by phytochromes throughout FR-HIR and VLFR responses.

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera.

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera. Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90-100 &mgr;mol m-2 s-1). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D-l-alpha-difluoromethylarginine (DFMA) and methylglyoxal bis(guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.

Effects of light quality on somatic embryogenesis in Araujia sericifera.

The effects of photoperiod, light quality and end-of-day (EOD) phytochrome photoconversion on somatic embryogenesis (SE) of Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 8- and 16-h photoperiods using Gro-lux fluorescent lamps. The photon fluence rate was 90-100 µmol m-2 s-1 and the red (R):far-red (FR) ratio was 98. R, FR, R followed by FR (R-FR) and FR followed by R (FR-R) light treatments were applied for 3 weeks at the end of the photoperiods. In a set of experiments, DL-alpha-difluoromethylarginine (DFMA) or methylglyoxal bis(guanylhydrazone) (MGBG), both inhibitors of polyamine biosynthesis, were added to the culture medium in order to study the involvement of polyamine metabolism. The level of SE was the same in long (LD) and short (SD) days. Thus, the light effect was accomplished after 8 h. All EOD treatments that decreased the Pfr level inhibited SE when applied after SD, but not after LD. The FR-R treatment after LD caused an additional stimulatory effect on SE, even in the presence of polyamine inhibitors. DFMA inhibited SE in both SD and LD, but MGBG did not modify SE in either SD or LD. The R, FR and R-FR treatments did not alter the level of SE when applied after LD in the presence of DFMA or MGBG. However, these treatments decreased SE after SD when the medium contained polyamine inhibitors. Our results suggest that Gro-lux lamps, which produce an extremely high R:FR ratio, promote SE in A. sericifera and a timing response to phytochrome photoconversion during photoperiodic induction. Thus, our data corroborate the involvement of phytochromes and polyamines in SE in A. sericifera, which responded as a light-dominant long-day plant.