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.     

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.     

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.     

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.     

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.     

Photomorphogenic effects of UV-B radiation on hypocotyl elongation in wild type and stable-phytochrome-deficient mutant seedlings of cucumber

Hypocotyl elongation responses to ultraviolet-B (UV-B) radiation were investigated in glasshouse studies of de-etiolated seedlings of a long-hypocotyl mutant ( lh ) of cucumber ( Cucumis sativus L.) deficient in stable phytochrome, its near isogenic wild type (WT), and a commercial cucumber hybrid (cv. Burpless). A single 6- or 8-h exposure to UV-B applied against a background of white light inhibited hypocotyl elongation rate by ca 50% in lh and WT seedlings. This effect was not accompanied by a reduction in cotyledon area expansion or dry matter accumulation. Plants recovered rapidly from inhibition and it was possible to stimulate hypocotyl elongation in plants exposed to UV-B by application of gibberellic acid. In all genotypes inhibition of elongation was mainly a consequence of UV-B perceived by the cotyledons; covering the apex and hypocotyl with a filter that excluded UV-B failed to prevent inhibition. These results indicate that reduced elongation does not result from assimilate limitation or direct damage to the apical meristem or elongating cells, and strongly suggest that it is a true photomorphogenic response to UV-B. The fact that UV-B fluences used were very low in relation to total visible light, and the similarity in the responses of lh and wild-type plants, are consistent with the hypothesis that UV-B acts through a specific photoreceptor. It is argued that, given the weak correlation between UV-B and visible-light levels in most natural conditions, the UV-B receptor may play an important sensory function providing information to the plant that cannot be derived from light signals perceived by phytochrome or blue/UV-A sensors.     

Effect of phytochrome activation followed by prolonged dark incubation on chloroplast development in etiolated cucumber cotyledons

The inhibitory effect of a short red light pulse followed by prolonged dark incubation on chlorophyll accumulation in etiolated cucumber cotyledons ( Cucumis sativus L. cv. Elem) was reflected in the development of the internal membrane system of the mesophyll plastids. Dark incubation for 24 h after phytochrome activation produced the characteristic accelerating effect OB chlorophyll synthesis and chloroplast development. However, longer intervening dark periods (48, 72 and 96 h) before white light exposure resulted not only in a diminished capacity to concentrate chlorophyll, but also in an impaired ability to form grana. The absence of stacking was consistent with a high chlorophyll a to chlorophyll b ratio.     

Absence of phyB inhibits hypocotyl elongation in dark-grown Ih cucumber seedlings: an active role for PrB

Cucumber (Cucumis sativus L.) seedlings carrying the long hypocotyl (Ih) mutation, which confers a lack of B-type phytochrome (phyB), were significantly shorter than their near-isogenic wild-type counterparts when grown in complete darkness. Relative growth rates determined for 5 mm hypocotyl regions were lower in Ih seedlings in all growing regions, and the zone of elongation was less extensive in Ih hypocotyls. Digital imaging microscopy revealed that the pattern of epidermal cell lengths along the stem axis differed between the Ih mutant and the iso-genic wild-type. These findings (and the fact that experiments were conducted under conditions where phytochrome photoconversion to the far-red-absorbing form does not occur) suggest that the red-absorbing form of phyB (PrB) is an active positive regulator of development in etiolated plants.     

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.     

Changes of polyamines and ethylene in cucumber seedlings in response to chilling stress

Cucumber ( Cucumis sativus L. cv. Victory) seedlings were exposed to chilling at 5°C and endogenous levels of polyamines and 1-aminocyclopropane-1-carboxylic acid (ACC) were measured after chilling and after warming at 20°C. The level of spermidine was higher in the chilled seedlings than in the non-chilled seedlings. Treatment with a plant bioregulator, (2RS,3RS)-1-(4-cholorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol (paclobutrazol), reduced the chilling injury and the levels of spermidine in the chilled seedlings. The levels of ACC and production of ethylene showed sharp increases after warming following exposure to chilling. These increases were suppressed by the application of aminooxyacetic acid (AOA). However, AOA treatment did not reduce chilling injury or affect the levels of polyamines in the tissue. These data indicate that the increase in ACC and ethylene is a response of the tissue to the chilling exposure and is not a cause of the injury. The data also suggest that the syntheses of polyamines and ethylene are not competitive with each other even under chilling stress conditions.     

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.     

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     

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     

Electrophysiological evidence for a role for calcium in temperature sensing by roots of cucumber seedlings

Abstract. Rapid-cooling pulses to non-stressful temperatures cause strong, transient depolarizations in cortical cells of cucumber roots. The amplitudes of these electrical responses are graded according to the rate and amplitude of the cooling pulse. Such graded potentials are typical of sensory processes and indicate that plants possess the ability to sense temperature change. La³⁺, a blocker of Ca²⁺ channels, and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-acetic acid (EGTA), a Ca²⁺ chelator, inhibit the electrical responses elicited by rapid-cooling pulses. High external [Ca²⁺] enhances them. These results indicate the involvement of a plasma membrane-associated Ca²⁺ channel in the process of temperature sensing by plants. Calmodulin antagonists prolong the repolarization phase of the electrical responses, suggesting a role for calmodulin in the recovery from stimulation.     

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.     

Effect of light on nucleotide modifications in the transfer RNA of cucumber cotyledons

The effect of light on nucleotide modifications in the tRNA of cucumber (Cucumis sativus L. var. Guntur) cotyledons was studied by chromatographic, electrophoretic and immunological methods. The tRNA from light-grown tissue showed the absence of 2-methylguanosine and a decrease in the relative proportions of ribothymidine and cytokinin-active ribonucleosides when compared to those produced from dark-grown tissue. On the other hand, a significant amount of one species of 2′-O-methyldinucleotide was observed in the tRNA of light-grown tissue which was not detected in the dark-grown tissue. Also, tRNA from light-grown tissue had higher levels of another species of 2′-O-methyldinucleotide. The results showed no difference in the amounts of other modified nucleosides in tRNA between tissues grown under the two conditions. 2′-O-Methyl-l-methyladenosine, a nucleotide modified both in the base and the ribose, apparently specific to plant tRNAs, has been found to be present in the RNA of both light- and dark-grown tissues. These results on the variation in modified nucleotides suggest that light has some role in nucleotide modification and, consequently, in cellular functions.     

Uptake and translocation of calcium in cucumber

Uptake and translocation of Ca²⁺(⁴⁵Ca) were compared with water translocation in 12-day old intact plants and excised roots of cucumber ( Cucumis sativus L. var. Cilla), which had been cultivated in nutrient solution. No immediate reduction of Ca²⁺ uptake was found when water translocation was reduced by excision of the shoot. In the presence of 2,4-dinitrophenol Ca²⁺ translocation was reduced in the intact plants while water translocation was unchanged. It is suggested that regulation of Ca²⁺ uptake is primarily achieved in the root. The DNP-sensitive mechanism of Ca²⁺ uptake was associated with the root and probably represented transport through the endodermis into the stele.     

亚适宜温光盐环境下油菜素内酯对黄瓜幼苗抗氧化系统及光合作用的影响

研究了24 表油菜素内酯(EBR)对亚适宜温光盐环境下黄瓜幼苗抗氧化系统及光合作用的影响.结果表明: 与对照相比,亚适宜温光盐环境下黄瓜幼苗叶片H₂O₂含量增加,膜脂过氧化程度加剧,膜透性增强,净光合速率(P_n)、气孔导度(g_s)、胞间CO₂浓度(C_i)和蒸腾速率(T_r)分别显著下降39.3%、40.0%、21.2%和47.2%,幼苗干物质积累减少35.9%.外源喷施EBR可提高亚适宜温光盐环境下黄瓜幼苗的抗氧化酶活性,降低H₂O₂含量及膜透性,缓解亚适宜温光盐环境下P_n、g_s、T_r的下降幅度,幼苗干物质积累增加25.9%,生长加快．EBR可通过调节亚适宜温光盐环境下黄瓜幼苗抗氧化性,减少其膜脂过氧化程度,进而维持其较高的光合性能,有效促进了亚适宜温光盐环境下黄瓜幼苗的生长．     

Mannitol inhibits growth of intact cucumber but not pea seedlings by mechanically collapsing the root pressure

The positive xylem pressure (Px) in cucumber hypocotyls is a direct extension of root pressure and therefore depends on the root environment. Solutions of the electrolyte KCl (0-10 osm) reduced the hypocotyl Px transiently (biphasic response), while the Px reduction by mannitol solutions was sustained. The amplitudes of the induced Px reduction depended directly, and the degree of Px restoration after stress release depended indirectly, on the size of the initial positive Px indicating that mannitol released the root pressure by a mechanical rather than osmotic mechanism. Mannitol treatment and other means of root pressure reduction revealed two separate growth responses in the affected cucumber hypocotyls. Only steep Px drops (following root excision or root pressure release in mannitol) directly cause a rapid, transient drop in growth rate (GR). Both rapid and slow (after root incubation in KCN or NEM) decreases in root pressure, however, led to a sustained growth inhibition of cucumber hypocotyls after about 30 min. This delay characterizes the growth response as an indirect consequence of the Px change. Pea seedlings, which lacked root pressure and had a negative Px throughout, showed extremely small changes in epicotyl Px and GR after root incubation in mannitol. It is apparent that the higher sensitivity of cucumber growth to mannitol depended on the presence and release of root pressure.