Impaired chlorophyll formation in etiolated cucumber cotyledons following prolonged dark incubation after red light pretreatment

Red light (R) pretreatment of etiolated cucumber seedlings ( Cucumis sativus L. var. Elem) followed by prolonged dark incubation prior to white light (WL) exposure, had an adverse effect on the greening of the cotyledons. The effect was photoreversible by far-red (FR) light. Cotyledons which were dark incubated for 24 h following the R pulse greened more rapidly when exposed to WL than did the controls, while total chlorophyll (Chl) accumulation after 24 h in the light was about the same in both. However, after 48 h post-R dark incubation greening of the treated cotyledons was delayed, and their amount of Chl which accumulated after 24 h WL was about one half of that in non-treated seedlings. As the length of the post-R dark incubation period was extended Chl production became slower, so that after 96 h post-R dark incubation the Chl level in the treated cotyledons after 24 h WL was approximately 20% of the controls. No significant differences in amounts of protochlorophyll could be detected between seedlings preilluminated with R or R followed by FR. Seedlings 4-, 5- and 6-days-old at the time of R treatment showed similar degrees of impaired Chl synthesis following prolonged post-R dark incubation.     

Mimicry by cytokinin of phytochrome-regulated inhibition of chloroplast development in etiolated cucumber cotyledons

A study of the kinetics of chlorophyll (Chl) synthesis in cotyledons of etiolated cucumber seedlings ( Cucumis sativus L . cv. Delilah) treated with 5×10^-5 M -ben-zyladenine (BA) showed that cytokinin, like a red light pulse, could inhibit as well as promote pigment accumulation depending on the length of the dark period following induction. Spraying intact, dark-grown seedlings with BA, 24 h prior to white light exposure, eliminated the lag phase in Chl synthesis, while treatment with hormone 72 h before greening not only delayed the onset of synthesis, but it also reduced the amount of Chl accumulated after 24 h continuous white light. Impairment of Chl formation was correlated with inhibited regeneration of protochlorophyll and delayed appearance of the light harvesting Chl alb polypeptide. Application of σ-aminolevulinic acid (15 m M ) 2 h before white light exposure shortened the lag phase in Chl synthesis in control as well as in inhibited cotyledons, but the adverse effect of the red light and BA treatments on long-term Chl accumulation (24 h) was not reversed. Application of glutamate did not stimulate Chl production. Simultaneous treatment with hormone and red light 72 h before greening enhanced their separate inhibitory effects on Chl synthesis, but when given together 24 h prior to white light, their promotive effects on pigment accumulation were not additive.     

Phytochrome-mediated development of glycine oxidation by mitochondria in cucumber cotyledons

The development of glycine oxidation activity in mitochondria in etiolated cucumber ( Cucumis sativus L., cv. Shinfushinari) cotyledons is regulated by phytochrome. This conclusion is based on two lines of evidence. 1. The oxidation activity was increased by continuous illumination of far-red light. 2. It was also increased by brief red light pulses, the effect of which was reversed by brief far-red light pulses. The light-induced increase in glycine oxidation and in glycine decarboxylase (EC 2.1.2.10) activity in the cotyledons was inhibited by cycloheximide, but not by chloramphenicol. While glycine oxidation activity continued to increase during light-illumination for 20 h, malate oxidation activity increased for 6 to 8 h after illumination and decreased thereafter. This transient increase in the activity of malate oxidation was also induced by red light pulses and the effect of the red light was reversed by far-red light pulses.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Effect of cinnamate on nitrate reductase activity in isolated cucumber cotyledons

The effect of cinnamic acid on in vivo nitrate reductase activity and protein content in cucumber cotyledons was studied. Cinnamate increased in vivo nitrate reductase activity and also the total protein content at lower concentrations (0.01–0.1 mM). Higher concentration, however, proved inhibitory. The effect of cinnamate on nitrate reductase activity has been discussed.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber ( Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.     

A two-fold action of benzyladenine on chlorophyll formation in etiolated cucumber cotyledons

Treatment of 3-day-old excised etiolated cotyledons of cucumber ( Cucumis sativus L. cv. Aonagajibai) with benzyladenine (BA) in the dark stimulates chlorophyll (Chl) formation during the lag phase (designated as 'lag elimination') and accelerates the steady-state rate of Chl formation under subsequent continuous illumination with white light. The separation of this two-fold effect is possible using two different methods of BA treatment in darkness: a brief BA treatment followed by various periods of water treatment in darkness, or various periods of continuous dark BA treatment. In either treatment, BA rapidly eliminates the lag phase (the fast-appearing effect) and after a longer time period accelerates the steady-state rate (the late-appearing effect). With a brief BA treatment, both effects decay rapidly. In contrast, with continuous BA treatment, none of the effects decay after reaching their maxima, particularly in cotyledons excised 2 days after sowing and aged for a long period before the onset of BA treatment. These facts indicate that BA acts as a trigger in stimulating Chl formation. The relationship between the actions of BA and light is discussed.     

Effect of manganese ions on benzyladenine-induced growth and chlorophyll synthesis in excised cucumber cotyledons

The behaviour of endogenous Mn²⁺ was studied by electron spin resonance spectro-scopy during benzyladenine-induced growth of excised cucumber ( Cucumis sativis L. cv. Long green) cotyledons. The level of endogenous Mn²⁺ was decreased by ben-zyladenine treatment, most pronounced after 96 h. MnCl₂ applied alone promoted chlorophyll synthesis at relatively low concentrations but in the presence of ben-zyladenine higher concentrations of MnCl₂ were required for stimulation of chlorophyll synthesis. A pronounced increase in growth was observed when Mn²⁺ was applied with benzyladenine at 96 h, when the decline in the endogenous level of paramagnetic Mn²⁺ was maximal.     

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 α 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.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

Effect of the hypocotyl hook on greening in etiolated cucumber cotyledons

Four-day-old etiolated cucumber cotyledons (Cucumis sativus, L.) were excised and allowed to green in white fluorescent light at 28 C. Cotyledons excised with a full hypocotyl hook exhibited a lag phase of 1 hour before entering the rapid greening phase, whereas cotyledons excised without any hypocotyl hook exhibited a lag phase of 6 hours. Cotyledons excised with varying lengths of hypocotyl hook accumulated chlorophyll roughly in proportion to the hook length. When cotyledons were excised with a full hook and were partially or totally shielded from light with aluminum foil, the samples with the hook covered accumulated more chlorophyll than the wholly exposed samples. The samples with the cotyledons covered showed no net accumulation of chlorophyll irrespective of hook's exposure to light. These data suggest the contribution of some factor or factors by the hypocotyl hook which reduce the lag phase during greening.     

Benzyladenine-induced stimulation of 5-aminolevulinic acid accumulation under various light intensities in levulinic acid-treated cotyledons of etiolated cucumber

Benzyladenine (BA) stimulated 5-aminolevulinic acid (ALA) accumulation in the presence of levulinic acid during illumination with 43 μmol m⁻² s⁻¹ light in excised etiolated cotyledons of cucumber ( Cucumis sativus L. cv. Aonagajibai). A short dark-pretreatment (6 h) with BA eliminated the lag phase of ALA accumulation. The rate of ALA accumulation during the steady-state phase in cotyledons pretreated with BA for a long period (14 h) was considerably accelerated compared to that in cotyledons pretreated with BA for 6 h. The rate of ALA accumulation during the lag phase was saturated at a very low light fluence (<1.4 μmol m⁻² s⁻¹) in both BA-pretreated and water-control cotyledons. The steady-state rate of ALA accumulation increased with increasing light fluence up to 43 μmol m⁻² s⁻¹ (parallel to that of Chl formation) in water-control cotyledons. In contrast, in cotyledons pretreated with BA for either 6 or 14 h, the steady-state rate reached a plateau at a very low light fluence. Based on the above results together with our finding that there are two components of Chl formation (M. Dei, 1984. Physiol. Plant. 62: 521–526) possible intermediate steps of Chl biosynthesis pathway affected by BA and light intensity are discussed.     

Effect of UV-B (290–320 nm) irradiation on growth and metabolism of cucumber cotyledons

Cucumber ( Cucumis sativus L. cv. Natsusairaku 3) seedlings were grown in a growth cabinet under UV-B (290–320 nm) irradiation (equivalent to the UV-B radiation normally incident at Tokyo, 36°N latitude, during clear sky conditions in mid-april on a weighted daily fluence basis) and a UV-B-free control condition. UV-B irradiation inhibited the growth of the cotyledons, i.e. the increase in area, and increase in fresh and dry weights of the cotyledons. The greatest inhibition rate was observed in the increase in area, causing a significant increase in specific leaf weight (the ratio of weight to area). UV-B irradiation had no significant effect on DNA and RNA contents in the cotyledons, but decreased protein content slightly. In contrast, the irradiation reduced the amounts of organic acids and soluble sugars, indicating that primary carbon metabolism was very sensitive to UV-B radiation. UV-B irradiation lowered the photosynthetic activity in the cotyledons without any effect on chlorophyll content and respiratory activity. These results indicate that UV-B radiation at the ambient level may act as a physiological stress in some UV-sensitive plants.     

Transient down-regulation of phytochrome mRNA abundance in etiolated cucumber cotyledons in response to continuous white light

Phytochrome mRNA abundance decreased to 20% of the initial level in etiolated cucumber (Cucumis sativus L.) cotyledons exposed to continuous white light. Unexpectedly, by 12 hours of continuous white light, phytochrome mRNA had reaccumulated to 60% of the control level. High stringency RNA blot analyses suggest that it is the mRNA encoding type I phytochrome that reaccumulates.     

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons

Benzyladenine-induced changes in the translatable mRNA population in excised cucumber cotyledons were studied. Poly (A)⁺ RNA was prepared from etiolated cotyledons incubated with or without benzyladenine (BA) for various periods in the dark. Using nonequilibrium pH gradient electrophoresis-SDS polyacrylamide gel electrophoresis and isoelectric focusing-SDS polyacrylamide gel electrophoresis, both basic and neutral proteins translated in vitro were separated. About 240 spots were detected and 16 of them changed within 6 h after BA application. Some spots changed quickly (within 1–2 h). Among them, three were repressed markedly     

Influence of electrolytes on growth, phototropism, nutation and surface potential in etiolated cucumber seedlings

A variety of electrolytes (10-30 mol m-3) increased the relative growth rate of etiolated cucumber (Cucumis sativus L. cv. Burpee's Pickler) hypocotyls by 20-50% relative to water-only controls. The nonelectrolyte mannitol inhibited growth by 10%. All salts tested were effective, regardless of chemical composition or valence. Measurements of cell-sap osmolality ruled out an osmotic mechanism for the growth stimulation by electrolytes. This, and the nonspecificity of the response, indicate that an electrical property of the solutions was responsible for their growth-stimulating activity. Measurements of surface electrical potential supported this reasoning. Treatment with electrolytes also enhanced nutation and altered the pattern of phototropic curvature development. A novel analytical method for quantitating these effects on growth was developed. The evidence indicates that electrolytes influence an electrophysiological parameter that is involved in the control of cell expansion and the coordination of growth underlying tropisms and nutations.     

外植体龄和蔗糖浓度对黄瓜子叶产生毛状根的影响

研究了外植体龄和蔗糖浓度对发根农杆菌 R160 1介导黄瓜子叶产生毛状根的影响。结果表明 :以 10 d龄子叶外植体产生毛状根的能力最强 ,外植体的毛状根诱导率为 88.89% ;2 0 d龄子叶外植体的毛状根诱导率比 10 d龄子叶外植体降低 52 .86% ;30 d龄子叶外植体感染发根农杆菌R160 1后不产生毛状根。感染发根农杆菌 R160 1的黄瓜子叶外植体在不加或加 1%蔗糖的 MS培养基上的毛状根诱导率极低 ,子叶外植体逐渐变黄 ,腐烂 ;而培养基中添加 2 % ,3%或 4 %的蔗糖可显著提高子叶外植体的毛状根诱导率。黄瓜毛状根能在无外源植物激素的 MS液体培养基中自主生长。冠瘿碱的高压纸电泳检测表明毛状根已被 Ri T- DNA转化     

Structural-functional changes in detached cucumber leaves, and modelling these by hormone-treated leaf discs

Senescence and rejuvenation were investigated in detached cucumber (Cucumis sativus L.) leaves after cultivation in nutrient solution for one week or four weeks. Rooting of the petiole (visible generally from the 7th day) elicited a combination of different morphological, anatomical, and physiological changes in the lamina. Extensive growth in area and thickness, extreme regreening, changes of chloroplast structure and activity, as well as the pattern of Chl-protein complexes were observed and compared either to the corresponding parameters of young detached leaves or mature attached leaves. These responses could be provoked separately by treating excised leaf discs with kinetin, benzyladenine, or indolylacetic acid. The hormones showed mutuality in their effects, benzyladenine being responsible for the growth of cells, while indolylacetic acid and kinetin promoted an increase in chlorophyll content. However, none of the treatments resulted in the growth of the chloroplasts in the leaf discs, which was only prominent in the rooting leaves.     

NADP+-isocitrate dehydrogenase in germinating cucumber cotyledons: Purification and characterization of a cytosolic isoenzyme

The activity of NADP⁺-dependent isocitrate dehydrogenase (ICDH, EC 1.1.1.42) was investigated during the post-germinative growth of cucumber ( Cucumis sativus L. cv. Marketmore) seedlings. Isoelectric focusing showed the presence of several isoenzymes, two of which represented 70–80% of the total NADP⁺-ICDH activity in cotyledons of seedlings grown in the dark. They had pI values between 4.8 and 5.8. The isoenzyme with higher pI was purified to homogeneity by hydrophobic interaction, affinity, hydroxylapatite and anion exchange chromatography. The purified isoenzyme is a dimeric protein, consisting of two apparently identical 43-kDa subunits. It is specific for NADP⁺, inhibited by ATP and by 2-oxoglutarate, whereas it is not inhibited by citrate, succinate, and glyoxylate. The data indicate that NADP⁺-ICDH from cucumber is structurally similar to ICDHs from other plants, but it shows some peculiar biochemical characteristics.