Accumulation of photosynthetic pigments in Larix decidua Mill. and Picea abies (L.) Karst. cotyledons treated with 5-aminolevulinic acid under different irradiation

European larch (Larix decidua Mill.) and Norway spruce [Picea abies (L.) Karst.] synthesize chlorophyll (Chl) in darkness. This paper compares Chl accumulation in 14-d-old dark-grown seedlings of L. decidua and P. abies after shortterm (24 h) feeding with 5-aminolevulinic acid (ALA). We used two ALA concentrations (1 and 10 mM) fed to cotyledons of both species in darkness and in continuous light. The dark-grown seedlings of L. decidua accumulated Chl only in trace amounts and the seedlings remained etiolated. In contrast, P. abies seedlings grown in darkness were green and had significantly higher Chl content. After ALA feeding, higher protochlorophyllide (Pchlide) content was observed in L. decidua than in P. abies cotyledons incubated in darkness. Although short-term ALA feeding stimulated the synthesis of Pchlide, Chl content did not change significantly in cotyledons incubated in darkness. The Chl accumulation in cotyledons fed with ALA was similar to the rate of Chl accumulation in the controls. Higher Chl accumulation was reported in control samples after illumination: 86.9% in L. decidua cotyledons and 46.4% in P. abies cotyledons. The Chl content decreased and bleaching occurred in cotyledons incubated with ALA in light due to photooxidation. Analyses of Chlbinding proteins (D1 and LHCIIb) by Western blotting proved differences between Chl biosynthesis in L. decidua and P. abies seedlings in the dark and in the light. No remarkable increase was found in protein accumulation (D1 and LHCIIb) after ALA application. Our results showed interspecific difference in Chl synthesis between two gymnosperms. Shortterm ALA feeding did not stimulate Chl synthesis, thus ALA synthesis was not the rate-limiting step in Chl synthesis in the dark.     

Role of Light in the Appearance of Glutamine Synthetase in Leaves of Pennisetum glaucum

Leaves of Pennisetum [Pennisetum glaucum (L) HHB 67] seedlings contained two isozymes of glutamine synthetase (GS, EC 6.3.1.2): cytosolic GS1 and chloroplastic GS2. Leaves of seedlings grown in light for seven days contained about twofold higher GS activity than etiolated leaves. In both light and dark grown seedlings, total GS, GS1 and GS2 activity declined with plant age with more pronounced effect in leaves of etiolated seedlings, and GS2 declined at a much faster rate than GS1. Exposure of etiolated seedlings to light markedly enhanced GS1 and GS2 activity. This increase in activity was not affected by cycloheximide, precluding light dependent de novo synthesis of the enzyme. Treatment of etiolated seedlings with photosynthetic inhibitor, dichlorophenyl dimethyl urea (DCMU) inhibited light dependent appearance of GS. Exogenous supply of sucrose to dark grown seedlings greatly increased the GS activity in dark. These results suggest that light-mediated stimulation in activity of GS in Pennisetum leaves is dependent on photosynthetic reaction.     

Diverse regulation by sucrose of enzymes involved in storage lipid breakdown in germinating lupin seeds

The regulatory function of sucrose in the activity of lipid-degrading enzymes was investigated in germinating seeds of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet). The study was conducted on isolated embryo axes, excised cotyledons and seedlings cultured in vitro for 96 h on medium with 60 mM sucrose or without the sugar. The activity of lipase (lipolysis), acyl-CoA oxidase and catalase (fatty acid β-oxidation) was enhanced in all studied organs cultured on medium without sucrose. The activity of cytosolic aconitase (glyoxylate cycle) was stimulated by sucrose in seedling axes and isolated embryo axes, whereas in seedling cotyledons and excised cotyledons, it was inhibited. The regulatory function of sucrose in phosphoenolpyruvate carboxykinase (gluconeogenesis) was observed only in isolated embryo axes and the activity was lower in carbohydrate deficiency conditions. The peculiar features of storage lipid breakdown in germinating lupin seeds and its regulation by sucrose are discussed.     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

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.     

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.     

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.     

Light activation of vindoline biosynthesis does not require cytomorphogenesis in Catharanthus roseus seedlings

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC) and desacetoxyvindoline 4-hydroxylase (D4H), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. The expression of TDC was restricted to the upper epidermis of cotyledons, whereas that of D4H was confined to laticifer cells. Light exposure of etiolated seedlings significantly induced D4H enzyme activity without changing the steady-state levels of D4H immunoreactive protein or modifying the cellular distribution of D4H expression in dark-grown seedlings. These results suggest that the early and late stages of vindoline biosynthesis occupy different cellular compartments, even in the early phases of etiolated seedling development. The role of light in activating the late stages of vindoline biosynthesis does not, therefore, seem to be related to the formation of the laticifer and idioblast cell types. It is concluded that light is not required for formation of these cell types, whereas regulatory factors, restricted to idioblasts and laticifers, may respond to light to activate localised expression of the late stages of vindoline biosynthesis.     

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.     

Effects of Temperature on the Pattern of Anthocyanin Accumulation in Seedlings of Polygonum cuspidatum

Polygonum cuspidatum seedling. Anthocyanin accumulated first in the lower part of hypocotyls and then the site of accumulation gradually extended toward the upper part of hypocotyls when seedlings were irradiated with white light (WL) at 25 C. Etiolated seedlings accumulated anthocyanin only in the upper parts (hook and cotyledons) when the seedlings were irradiated with WL at 5 C. De-etiolated seedlings that had been pre-irradiated with WL for 1 day at 25 C accumulated anthocyanin both in upper and lower parts of the seedlings when the seedlings were irradiated with WL at 5 C. Spectral sensitivity was dependent on the temperature during irradiation. Red light (R), blue light (B), and near ultra-violet light (NUV) induced the accumulation of anthocyanin at 5 C but only NUV was effective in inducing the accumulation of anthocyanin at 25 C. Dichlorophenyl dimethylurea (DCMU) inhibited WL-induced anthocyanin accumulation but did not NUV-induced anthocyanin accumulation at 25 C. However, sucrose promoted NUV action at 25 C, indicating that photosynthesis can promote NUV-induced anthocyanin accumulation. Distribution of phytochrome in etiolated seedlings, that was examined by spectrophotometry, was similar to the distribution of anthocyanin at 5 C. Furthermore, phytochrome remained after 48 hr irradiation with WL at 5 C although phytochrome was rapidly degraded at 25 C. Received 12 July 1999/ Accepted in revised form 24 December 1999     

Stimulation of cytokinins and chlorophyll synthesis in cucumber cotyledons by triadimefon

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)-2-butanone] changes the morphology and partitioning of dry matter in cucumber ( Cucumis sativus L. cv. National Pickling) seedlings. The dry weights, potassium and cytokinin levels in the cotyledons and roots of the treated seedlings were higher, whereas the hypocotyl weights were lower than the controls. When etiolated intact seedlings or cotyledons excised from triadimefon-pretreated dark-grown seedlings were exposed to light, chlorophyll synthesis in the pretreated cotyledons was stimulated. Triadimefon does not have cytokinin-like activity in the cucumber cotyledon greening bioassay, but appears to induce the plants to produce more cytokinims, probably by stimulating root growth. Hence it is proposed that the stimulation of chlorophyll production by triadimefon in cucumber cotyledons is mediated by maintaining high levels of potassium and cytokinins in the cotyledons.     

Role of leaves in phototropism

Experiments with green seedlings of sunflower (Helianthus annuns L.) indicate the existence of a phototropic mechanism which involves the leaves or cotyledons, and which can produce an asymmetry of auxin content without the involvement of lateral auxin transport, the classic explanation of phototropism in etiolated seedlings. The basic lines of evidence for the leaf-mediated tropism are: 1) darkening of one cotyledon will cause curvature of the stem toward the lighted cotyledon: 2) the darkened cotyledon sustains an enhanced growth rate in the stem below it: 3) conversely, light suppresses the growth-stimulating effects of a single cotyledon: and 4) more diffusible auxin is obtained from the stem below darkened cotyledons than below lighted ones.     

Light Induced Enhancement in Proline Levels Under Stress is Regulated by Non-Photosynthetic Events

Vigna radiata (L.) seedlings (5-d-old) were exposed to different concentrations of NaCl in light and in dark. The content of proline in the shoots increased with an increase in NaCl concentration, in light as well as in dark. But, irrespective of the concentration of NaCl, proline accumulation in the shoots was higher in light than in dark. Pretreatment of seedlings with dichlorophenyl dimethyl urea (DCMU) did not make any significant difference in light promoted stress induced proline accumulation. As DCMU is a potent inhibitor of photosynthetic electron transport, the light reaction of photosynthesis was not responsible for the observed light promotion of stress induced proline accumulation. In another set of experiments, 5-d-old green as well as etiolated seedlings were exposed to NaCl stress in the presence of different concentrations of sucrose. Irrespective of the concentration of sucrose used, proline content in shoots of stressed seedlings was higher in light than in dark. Although, sucrose enhanced NaCl stress induced increase in proline content in dark by about 32 %, this enhancement was not comparable to the 286 % increase in proline content brought about by light. These results showed that certain factors other than photosynthesis play a role in light promotion of stress induced proline accumulation.