Auxin biogenesis: subcellular compartmentation of indoleacetaldehyde reductases in cucumber seedlings

Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldehyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPH-specific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomal fraction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subcellular compartments.     

Chloroplast Biogenesis 34: SPECTROFLUOROMETRIC CHARACTERIZATION IN SITU OF THE PROTOCHLOROPHYLL SPECIES IN ETIOLATED TISSUES OF HIGHER PLANTS

The fluorescence emission and excitation properties of protochlorophyll in etiolated cucumber (Cucumis sativus L.) cotyledons and primary bean (var. Red Kidney) leaves were characterized at 77 K. Contrary to previous studies, it appears that the short-wavelength protochlorophyll emission band consists of four fluorescent components, instead of only one nonphototransformable protochlorophyll. It was demonstrated that etiolated cucumber cotyledons synthesize and accumulate nontransformable protochlorophyll (E₄₄₀, F₆₃₀) as well as short-wavelength phototransformable protochlorophyll (E₄₃₃, F₆₃₃), (E₄₄₄, F₆₃₆), and (E₄₄₅, F₆₄₀). Long-wavelength phototransformable protochlorophyll (E₄₅₀, F₆₅₇) is also formed. In this context, E refers to the Soret excitation maxima and F refers to the red emission maxima of the protochlorophylls.     

The role of indoleacetaldehyde in IAA production from tryptophan by plants and by their epiphytic bacteria

Tryptophan, tryptamine, or indolepyruvic acid were applied to 2 systems: a bacterial (pea stem sections containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In the plant system, the production of indoleacetic acid and indoleethanol (tryptophol) from each applied indole derivative is clearly reduced by the aldehyde reagents bisulfite and dimedon, respectively. Indoleacetaldehyde is chromatographically detected after alkaline liberation from its bisulfite addition product. In the bacterial system, the production of indoleacetic acid and indoleethanol is likewise reduced by bisulfite and dimedon. However, after tryptophan or tryptamine application, we could not detect indoleacetaldehyde in the described way. In one case only, namely tryptamine application to the bacterial system, indoleethanol production (contrary to indoleacetic acid production) is scarcely reduced by the aldehyde reagents. This indicates a bacterial pathway tryptamine → indoleethanol which bypasses indoleacetaldehyde.     

Synthesis and cytokinin-like activity of 7-chloro-imidazo[1,2-c]pyrimidines

Some 5-amino substituted 7-chloro-imidazo[1,2-c]pyrimidines and 7-chloro-8-methylthio-imidazo[1,2-c]pyrimidines were synthesized for further information on the role of the purine ring in cytokinin structure/activity relationships. Their cytokinin-like activity was examined using four different tests: expansion of cucumber etiolated cotyledons; formation of chlorophyll in etiolated cotyledons of cucumber; preservation of chlorophyll breakdown in sections of barley leaves; inhibition of root growth in intact wheat seedlings. Compounds with the imidazopyrimidine ring were generally less active than those with the purine ring, with the exception of the pentenylamino derivatives, which showed an activity comparable with that of the control, kinetin.     

Metabolism of Indole-3-acetaldehyde.

Aerobic infiltration of synthetic indoleacetaldehyde (IAAld) in buffered medium of pH 4.55, into living tissues of lower and higher plants, leads in the majority of cases to the formation of both indoleacetic acid and tryptophol. This activity is evinced by etiolated as well as green tissues. Besides, all parts of higher plants tested — roots, cotyledons, hypocotyls and leaves possess this activity. Abolishment of this activity by boiling indicates its enzymic nature. Coupled with the established occurrence of indoleacetaldehyde, the widespread distribution of such activity, strengthens the probability that indoleacetaldehyde may be the normal and natural precursor in the biosynthesis of indoleacetic acid.     

Influence of chloroplast development on the activation of the diphenyl ether herbicide acifluorfen-methyl

The activity of acifluorfen-methyl (AFM); methyl 5-(2-chloro-4-[trifluoromethyl] phenoxy)-2-nitrobenzoate in excised cucumber cotyledons (Cucumis sativus L.) was examined. AFM induced membrane disruption, was significantly greater when etiolated cotyledons were illuminated 16 hours at 150 microeinsteins per square meter per second photosynthetically active radiation versus incubation under illumination of 4-fold greater intensity. These results were unexpected since the loss of membrane integrity is initiated by photodynamic reactions. Untreated, etiolated cotyledons were not able to accumulate chlorophyll under the higher light intensity while control and herbicide treated cotyledons greened significantly under the lower intensity illumination suggesting that some process associated with greening stimulated AFM activity. Inhibition of greening by cycloheximide also reduced AFM activity. Intermittent lighting induced greening in AFM treated cotyledons without causing any detectable loss of plasmalemma integrity. Utilization of this system for pretreatment of cotyledons prior to continuous illumination revealed that activity was greater when tissue was greened in the presence of AFM than when herbicide treatments were made after a greening period of the same duration. The results indicate that the pigments in situ in etiolated tissue are sufficient, without greening, to initiate membrane disruption by AFM. However, greening increases the herbicidal efficacy greatly. Furthermore, the stimulation appears to be due to specific interactions between AFM and the developing plastid and is not attributable solely to an increase in endogenous photosensitizers.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.     

Chlorophyll determination in intact tissues using n,n-dimethylformamide

Photosynthetic pigments from etiolated cucumber (Cucumis sativus var. Beit Alpha improved, Hazera Co., Gedera) cotyledons were extracted by direct immersion of the intact cotyledons into the solvent N,N-dimethylformamide (DMF). The solvent is especially efficient when pigment concentration is low; time and tools are saved and the loss of pigment that usually occurs in more complicated extraction procedures is prevented. The specific absorption coefficient of chlorophyll a in DMF was also determined.     

Pathways of IAA production from tryptophan by plants and by their epiphytic bacteria: Metabolism of indolepyruvic acid and indolelactic acid

Metabolites of indolepyruvic acid and indolelactic acid were investigated using 2 systems: a bacterial (pea stem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The products of spontaneous indolepyruvic acid decomposition in aqueous solution and during chromatography were investigated, too. Biological indolepyruvic acid conversion yields, besides those substance amounts which occur spontaneously, indoleacetic acid, indoleethanol (tryptophol) and (only in the sterile plant system) indoleacetaldehyde. An inhibitor extract from pea stems decreases the indoleacetic acid and increases the indoleethanol and indoleacetaldehyde gain. Indolelactic acid is not metabolized in the sterile plant sections. Indolelactic acid oxidation by the bacteria-containing homogenate yields indolepyruvic acid and is inhibited by the inhibitor extract.     

The effect of gibberellic acid on phytochrome measurement in vivo

Phytochrome changes in hypocotyls of light grown mustard seedlings (Sinapis alba L.) and the phytochrome destruction in cotyledons of etiolated plants have been analysed as influenced by exogenous gibberellic acid (GA₃). The rate of phytochrome appearance in hypocotyls was decreased after GA₃ treatment. Destruction of phytochrome in cotyledons of etiolated seedlings was significantly accelerated by the same hormone treatment. This effect was also observed in the hypocotyls of light grown seedlings.     

Stimulation by Ethylene of Chlorophyll Biosynthesis in Dark-grown Cucumber Cotyledons

Five-day-old etiolated cucumber (Cucumis sativus L. var. Alpha Green) cotyledons produced more chlorophyll over a 4-hour illumination period after a prolonged exposure (12 to 72 hours) in the dark to ethylene concentrations ranging from 0.1 to 10 μl/l. Intact seedlings and excised cotyledons responded in the same way to this treatment. This effect does not involve a shortening of the lag phase of chlorophyll accumulation. Exposure of cotyledons to ethylene during the illumination period did not produce the same stimulatory effect on chlorophyll synthesis and, under certain conditions, chlorophyll synthesis was slightly inhibited by exposure to ethylene in the light.     

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.     

Phytochrome Photoconversion in Vivo: Comparison between Measured and Predicted Rates

The measured rates of phytochrome photoconversion in vivo, in etiolated cabbage (Brassica oleracea L.) seedlings and cucumber (Cucumis sativus L.) cotyledons, under blue, red, and far red irradiation, are significantly different from those predicted on the basis of the spectral photon flux distributions of the light sources and optical parameters of purified phytochrome. The geometrical relationships between the light source and the irradiated sample affect the rate of phytochrome photoconversion, which is significantly faster in cabbage seedling laying flat on white, wet filter paper than in seedlings in a vertical position. Light reflected from the white filter paper on the bottom of the dish contributes significantly to phytochrome photoconversion. Substituting the white filter paper with a less reflective black one results in a significant decrease of the rate of phytochrome photoconversion in cucumber cotyledons.     

Mg-protoporphyrin-IX and delta-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. Mg-protoporphyrin-IX synthesis

A crude plastid preparation from greening cucumber cotyledons was able to accumulate Mg-protoporphyrin-IX when incubated in the presence of glutamate. ¹⁴C from l-[U-¹⁴C]glutamate was incorporated into the porphyrin. The product was identified by its emission and excitation fluorescence spectra and by its chromatographic behavior on cellulose thin layers. The biosynthesis had a marked requirement for ATP and O₂. α,α′-Dipyridyl, a metal ion-chelating agent which had been shown to stimulate the synthesis of Mg porphyrins and phorbins in etiolated bean leaves and other whole tissues, stimulated likewise the in vitro synthesis of Mg-protoporphyrin-IX by isolated greening chloroplasts. Freezing and thawing, which destroy organelle integrity, abolished the ability of these isolated plastids to biosynthesize Mg-protoporphyrin-IX.     

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.     

Inter-organ Control of Greening in Etiolated Cucumber Cotyledons

Inter-organ control of greening in etiolated cucumber (Cucumis sativus L. cv. Aonagajibae) cotyledons was investigated. Four- or six-day-old excised or intact etiolated cucumber cotyledons were illuminated under aerobic conditions. Excised cotyledons without hypocotyl hooks produced chlorophyll without a prolonged lag phase and the rate of chlorophyll formation was not depressed if they were illuminated immediately after excision. If the excised cotyledons were incubated in the dark before illumination, chlorophyll accumulation at the end of 6 h of continuous illumination was remarkably lowered as the dark period lengthened, especially in 6-day-old cotyledons. The rapid loss of chlorophyll-forming capacity of excised cotyledons during dark preincubation suggests a stimulatory effect of hypocotyls on the greening in cotyledons. The treatment of excised cotyledons with bleeding sap in the dark for 18 h resulted in the promotion of chlorophyll formation during subsequent continuous illumination. Partial fractionation of bleeding sap with organic solvents and paper chromatography indicates that the active substances showed the same behavior as cytokinins. These facts add weight to the hypothesis that cytokinins from roots flow into cotyledons and stimulate greening.     

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.     

Indole-3-ethanol Oxidase: Kinetics, Inhibition, and Regulation by Auxins

We report the further characterization of indole-3-ethanol oxidase from cucumber seedlings. The effects of various inhibitors suggest that the enzyme may be a flavoprotein with a metal ion and sulfhydryl groups required for full activity. Indole-3-acetaldehyde, a product of the reaction, inhibits the enzyme. This inhibition is overcome by O₂ but not by indole-3-ethanol, indicating that the kinetic mechanism of the enzyme is a ping-pong Bi-Bi. The enzyme undergoes cooperative interactions with indoleethanol, yielding Hill coefficients as high as 2.96. Gibberellins are without effect on the enzyme, but it is inhibited by several acidic indoles possessing growth-promoting activity and by two synthetic auxins, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. Increasing concentrations of indoleacetic acid (IAA) brought about a slight reduction in the indoleethanol concentration producing halfmaximal velocity. Increasing levels of indoleethanol decreased the concentration of IAA required for half-maximal inhibition. At low concentrations of indoleethanol, low levels of IAA activated rather than inhibited. The effect of IAA was not overcome at higher levels of indoleethanol. These results may be interpreted as showing that IAA is a noncompetitive inhibitor which binds to that conformation of the enzyme which also binds indoleethanol. The significance of these interactions for the regulation of IAA biosynthesis is discussed.     

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.     

Development of glutamate:glyoxylate aminotransferase in the cotyledons of cucumber (Cucumis sativus) seedlings.

Glutamate:glyoxylate aminotransferase had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration (Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. Glutamate:glyoxylate aminotransferase activity was already present in the etiolated cotyledons of cucumber (Cucumis sativus) seedlings, and increased during greening. The enzyme was present only in the cytosol of the etiolated cotyledons and appeared in the peroxisomes during greening. The enzyme was purified to homogeneity from the cytosol of the etiolated cotyledons and from the peroxisomes of the green cotyledons of cucumber seedlings. The two enzyme preparations had nearly identical enzymic and physical properties. On the basis of these findings, roles of glutamate:glyoxylate aminotransferase in the glycollate pathway in photorespiration, and the mechanism of its appearance in the peroxisomes during greening, are discussed.