Gibberellin-Induced Suppression of Chloroplast Starch Formation from Exogenous Sucrose in Isolated Epidermis of Light-Grown Cucumber Hypocotyls

The effects of gibberellin A₃(GA₃) on chloroplast starch formationfrom exogenous sucrose in epidermal strips of light-grown cucumber(Cucumis sativus L. cv. Aonagajibai) hypocotyls were studied.Chloroplast starch formation was measured by counting microscopicallythe number of I₂KI stained chloroplasts. In the presence ofsucrose (1–50 mM) and in light, chloroplast starch formationoccurred rapidly for 5 hr and then the rate declined. GA₃ (100µM) simultaneously supplied with sucrose clearly suppressedboth the rate and the degree of starch formation. When the epidermiswas preincubated in the dark with sucrose in the presence orabsence of GA₃ and then post-incubated in light with bufferonly in the presence or absence of GA₃, starch formation wassuppressed to the same degree by GA₃ given either in the pre-or post-incubation period. When the epidermis was preincubatedin the dark with GA₃ alone and then transferred to light forpost-incubation with sucrose alone, GA₃ suppressed the initialrate of starch formation during the post-incubation period.The degradation of chloroplast starch formed in advance fromexogenous sucrose in light was not significantly affected byGA₃ These results are discussed in relation to the mechanismof the GA₃ action and also to the GA₃-induced decrease in theosmotic potential of the cell. (Received February 2, 1982; Accepted May 31, 1982)     

Gibberellin-Induced Changes in the Water Absorption, Osmotic Potential and Starch Content of Cucumber Hypocotyls

The effects of GA₃ on the water absorption, osmotic potentialand starch content of light-grown cucumber (cv. Aonagajibai)hypocotyl sections were examined. GA₃ (100µM.) stimulatedfresh weight increase as well as elongation. It had no effecton the dry weight change in the presence, or absence, of 50mM sucrose although dry weight increased significantly in thepresence of sucrose. In the absence of sucrose the weight wasunchanged. The osmotic potential of the epidermal cells of sectionsincubated with GA₃ was lower than that of the control, and thedifference between the two values was larger in young seedlings.When sections were incubated with sucrose, the osmotic potentialgreatly decreased. This decrease was more marked in the presenceof GA₃. GA₃ reduced the starch content of the sections bothin the presence and absence of sucrose. The total amount ofstarch, however, was markedly increased in its presence. Thedegradation of starch formed in advance from exogenous sucrosein light was not accelerated by GA₃. We discuss a possible rolefor gibberellin in cell elongation, based on our results, interms of cell water relations. (Received January 18, 1983; Accepted July 19, 1983)     

Growth regulation,plastid differentiation and the development of a photosynthetic system in cultured carrot root explants as influenced by exogenous sucrose and various phytohormones

Chromoplasts, which exist in the cells of freshly isolated carrot root explants, seemed to be transformed in thylakoid containing plastids, and chlorophyll formation was initiated if the explants were cultured in a liquid medium containing inositol and IAA as a hormonal supplement. This process was intensified when kinetin was also added, but no dependence on a sucrose supply could be found. A sucrose supply of 2% in conjunction with the combination of all three hormones, however, was needed to achieve maximal thylakoid formation including stacking in individual chloroplasts and for the very extensive chloroplast multiplication in explants growing with high cell division activity. It should be noted that the number of plastids per cell is strongly increased by the sucrose supplement which leads also to starch accumulation. However, no transformation into chloroplasts occurred without the hormonal stimulus.     

Pigment Degradation in Discs of the Thermophilic Cucumis sativus as Affected by Light, Temperature, Sugar Application and Inhibitors

Chlorophyll degradation in Cucumis leaf discs was measured at different temperatures between 1 and 25°C in the light and in darkness, and in the presence or absence of sucrose. Two different processes of chlorophyll degradation could be distinguished, a light-requiring process operating at 1 and 5°C and another, light and sucrose enhanced degradation process which was evident at 25°C. Degradation of leaf pigments at low temperatures was of a photo-oxidative nature since there was no degradation in the dark. The chlorophyll a/b ratio was decreased, carotene was degraded at a faster rate than chlorophyll, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and triphenyltetrazolium chloride (TTC) which prevent photo-oxidation, protected against chlorophyll degradation. The light and sucrose enhanced chlorophyll degradation at 25°C was of an enzymatic nature since it occurred in the dark as well as in the light. The chlorophyll a/b ratio was not affected, and carotene and chlorophyll degradation occurred at the same rate. Since DCMU completely inhibited the light enhancement at 25°C and experimentation in a low oxygen atmosphere also protected chlorophyll against the effect of light and sugar application, it is suggested that the enhancement of chlorophyll degradation by light and sucrose at 25°C may be due to increased sugar uptake of the chloroplasts and consequently excessive starch formation in the organelles.     

The role of the osmotic potential of the cell in auxin-induced cell elongation

IAA-induced elongation of light-grown cucumber hypocotyl sectionswas examined with respect to the osmotic relationship of thecell. Sucrose suppression of IAA-induced elongation in the lightoccurred at a lower sucrose concentration than in the dark,but there was no difference in the mannitol concentration whichsuppressed elongation. This differential sucrose suppressioncould be explained by the difference in the osmotic potentialof the cells between light and darkness. It was lower in thedark than in light, and the difference was more distinct inthe presence of sucrose. Treatment of sections with a photosyntheticinhibitor, CMU, also resulted in the maintenance of a low osmoticpotential. Under the experimental conditions where a largerIAA-induced elongation was obtained, a lower osmotic potentialwas also obtained. The results are discussed with respect tothe role of the osmotic potential of the cell in the enhancementof IAAinduced elongation. (Received April 3, 1978; )     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation, in kiwifruit culturedin vitro

Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells under normal Mg concentration (35 mg l⁻¹), and positively under high Mg concentration (105 mg l⁻¹ ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate. Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose.     

Die Bedeutung des Glucose-1-Phosphates für die Stärkesynthese in den Bündelscheidenzellen von Zea mays

Summary After illumination intact leaves of Zea mays contain sucrose and starch. The latter is located mainly in the bundle sheath cells. When 0.5 mm wide leaf strips are incubated with sucrose solution, the starch deposit in the bundle-sheath chloroplasts is greatly increased by light. When isolated bundle sheath cells are suspended in water or solutions of sucrose and various metabolites they are not capable of synthesizing starch. An appreciable production of starch in the chloroplasts of isolated bundle sheath cells can be observed only in the presence of glucose-1-phosphate.     

Effects of different planting dates and low light on cotton fibre length formation

Declining temperature and low light often appear together to affect cotton (Gossypium hirsutum L.) growth and development. To investigate the interaction on fibre elongation, two cultivars were grown in fields in 2010 and 2011 and in pots in 2011 under three shading levels for three planting dates, and the differences of environmental conditions between different planting dates were primarily on temperature. Fibre length in the late planting date 25 May was the longest instead of the normal planting date. Late planting prolonged fibre elongation period and the effect of late planting on fibre length formation was greater than low light. In the normal planting date, shading increased fibre length through delaying the peak of β-1,3-glucanase gene expression and bringing the peak of β-1,3-glucan synthase gene expression forward, leading to a longer duration of plasmodesmata(PD) closure to increase fibre length, instead of changing sucrose contents or relate enzyme activities. However, in the late planting dates, the difference of the duration of PD closure between shading treatments was not obvious, but low light had a negative impact on sucrose contents, sucrose synthase (SuSy) and vacuolar invertase(VIN) activities during fibre rapid elongation period, leading to the decline of fibre length. Due to late planting and low light, the decreased extent of fibre length of Sumian 15 was larger than Kemian 1. Under the combined condition, Sumian 15 had a shorter gene expression of Expansin, and more sensitive sucrose content, VIN and SuSy activity during fibre rapid elongation period. This resulted in the length formation of Sumian 15 which was more sensitive than Kemian 1, when the cotton suffered the combined effects.     

Light-dependent development of single cell C4 photosynthesis in cotyledons of Borszczowia aralocaspica (Chenopodiaceae) during transformation from a storage to a photosynthetic organ

BACKGROUND AND AIMS: Previous work has shown that Borszczowia aralocaspica (Chenopodiaceae) accomplishes C4 photosynthesis in a unique, polarized single-cell system in leaves. Mature cotyledons have the same structure as leaves, with chlorenchyma cells having biochemical polarization of dimorphic chloroplasts and C4 functions at opposite ends of the cell. KEY RESULTS: Development of the single-celled C4 syndrome in cotyledons was characterized. In mature seeds, all cell layers are already present in the cotyledons, which contain mostly lipids and little starch. The incipient chlorenchyma cells have a few plastids towards the centre of the cell. Eight days after germination and growth in the dark, small plastids are evenly distributed around the periphery of the expanding cells. Immunolocalization studies show slight labelling of Rubisco in plastids in seeds, including chlorenchyma, hypodermal and water storage, but not epidermal, cells. After imbibition and 8 d of growth in the dark labelling for Rubisco progressively increased, being most prominent in chlorenchyma cells. There was no immunolabelling for the plastid C4 enzyme pyruvate, Pi dikinase under these conditions. Cotyledons developing in light show formation of chlorenchyma tissue, induction of the cytosolic enzyme phosphoenolpyruvate carboxylase and development of dimorphic chloroplasts at opposite ends of the cells. Proximal chloroplasts have well-developed grana, store starch and contain Rubisco; those located distally have reduced grana, lack starch and contain pyruvate, Pi dikinase. CONCLUSIONS: The results show cotyledons developing in the dark have a single structural plastid type which expresses Rubisco, while light induces formation of dimorphic chloroplasts from the single plastid pool, synthesis of C4 enzymes, and biochemical and structural polarization leading to the single-cell C4 syndrome.     

OSMOTIC EFFECT ON THE PHOTOSHRINKAGE OF CHLOROPLASTS

Shrinkage of spinach chloroplasts by illumination and that byexposure to a high tonicity raised by addition of sucrose wereinvestigated by means of the rapid measurement of chloroplastvolume with a COULTER counter. The osmotic shock in the darkinduced two steps of volume change; almost instantaneous shrinkagefor the osmotic shock and follow-up gradual swelling. The finalvolume attained after equilibration was smaller than the originalvolume below 0.6 M, and greater above this concentration. Whenchloroplasts under osmotic shock were illuminated, the photoshrinkagecompeted with the swelling induced by the osmotic shock, andthese reverse effects were balanced at a certain volume. Photoshrinkageactivity measured after equilibration decreased with increasingconcentrations, and the activity curve plotted against sucroseconcentration showed a stationary level of 50% of the originalactivity between 0.2 and 0.6 M, indicating the resistance ofa structure in chloroplasts to the denaturation by the osmoticeffect of sucrose. The osmotic effect in the dark as well asin the light was completely reversible below 0.2 M and was partiallyreversible or irreversible above this concentration. Glucoseshowed qualitatively the same osmotic effect as sucrose. ¹ Present address: Laboratory of Chemistry of Natural Products,Tokyo Institute of Technology, Meguroku, Tokyo.     

Light-Induced Chloroplast Differentiation in Soybean Cells in Suspension Culture : Ultrastructural Changes during the Bleaching and Greening Cycles

Suspension cultures of SB-P cells of soybean (Glycine max) provide a novel, reproducible, and readily manipulable greening system useful for inducing chloroplast differentiation. The cells are subcultured and grown heterotrophically (3% sucrose) in the dark for at least three successive 14-day periods, subcultured and grown in the dark for 7 days more, and finally placed under white light and grown photoautotrophically. Chlorophyll begins to accumulate by 1 hour of light and continues up to 12 days. The chlorophyll a:chlorophyll b ratio is 3:1. Dark-grown cells contain a small amount of total carotenoids which increase 10-fold during greening. Chloroplast differentiation is strictly light dependent, with photosynthetic pigments accumulating in the light and being lost from cells returned to the dark. In the dark, the chloroplasts dedifferentiate to amyloplasts as the organized thylakoid network is lost and starch accumulates. Under continuous light, the amyloplasts differentiate into mature chloroplasts as the organelle elongates, becomes spanned by several bands of thylakoids, and undergoes grana formation. Chloroplast differentiation in SB-P cells is similar to that in intact angiosperms developing under normal light-dark cycles.     

Effect of NaCl,Na<Subscript>2</Subscript>SO<Subscript>4</Subscript>, and mannitol on utilization of storage starch and formation of plastids in the cotyledons and roots of alfalfa seedlings

Utilization of storage starch in the cells of cotyledon mesophyll and root meristem in the course of alfalfa (Medicago sativa L.) seed germination on the solutions of NaCl, Na₂SO₄, and mannitol at different concentrations and identical osmotic pressure was investigated using the method of transmission electron microscopy. Ultrastructural analysis showed changes in the number of starch grains and deceleration of chloroplast development depending on the osmotic component of salt influence. At low concentrations corresponding to osmotic pressure of 202.6 kPa, Na₂SO₄ did not affect the formation of the photosynthetic machinery and utilization of starch inclusions; mannitol contributed to the preservation of considerable reserve of starch without disturbing the development of chloroplasts; NaCl did not inhibit the development of the photosynthetic machinery and induced an increase in the number of starch grains presumably at the expense of newly produced starch. When the concentration of the investigated substances increased up to the values corresponding to the osmotic pressure of 607.8 kPa, NaCl did not suppress transformation of amyloplasts into chloroplasts and utilization of starch; Na₂SO₄ inhibited the development of chloroplasts and starch utilization; mannitol decelerated transformation of amyloplasts and inhibited mobilization of starch grains. The obtained results make it possible to propose a method of preliminary estimation of tolerance of dicotyledons to abiotic stresses based on the cytological analysis of utilization of starch grains and formation of photosynthetic compartments of chloroplasts in the mesophyll of cotyledons.     

Characterization of diurnal changes in activities of enzymes involved in sucrose biosynthesis

Experiments were conducted with vegetative soybean plants (Glycine max [L.] Merr., `Ransom') to determine whether the activities in leaf extracts of key enzymes in sucrose metabolism changed during the daily light/dark cycle. The activity of sucrose-phosphate synthase (SPS) exhibited a distinct diurnal rhythm, whereas the activities of UDP-glucose pyrophosphorylase, cytoplasmic fructose-1,6-bisphosphatase, and sucrose synthase did not. The changes in extractable SPS activity were not related directly to photosynthetic rates or light/dark changes. Hence, it was postulated that the oscillations were under the control of an endogenous clock. During the light period, the activity of SPS was similar to the estimated rate of sucrose formation. In the dark, however, SPS activity declined sharply and then increased even though degradation of starch was linear. The activity of SPS always exceeded the estimated maximum rate of sucrose formation in the dark. Transfer of plants into light during the normal dark period (when SPS activity was low) resulted in increased partitioning of photosynthate into starch compared to partitioning observed during the normal light period. These results were consistent with the hypothesis that SPS activity in situ was a factor regulating the rate of sucrose synthesis and partitioning of fixed carbon between starch and sucrose in the light.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

The Relationship of Carbohydrates to Cold Acclimation of Hedera helix L. cv. Thorndale

While both the total sugar content and cold hardiness greatly increased during artificial cold acclimation, no direct parallelism was demonstrated. In fact, plants hardened in the dark exhibited an increase in hardiness during the period when the total sugar content declined, furthermore, while there was evidence for an accumulation of sugars (especially sucrose) incorporation of ¹⁴C indicated that all of the isolated fractions were in a dynamic state. Dehardening, paralleled by a large increase in the starch content, was more rapid in the light than in the dark. Furthermore, in stems the sugar content increased after 3 days of dehardening but declined after 7 days. This increase may represent the release of sugars from a previously unextractable form such as a glycoprotein complex. Starvation experiments indicate that photosynthates produced during the cold acclimation period are preferentially used during cold acclimation rather than reserve carbohydrates. This was also indicated by the smaller amount of starch hydrolysis in plants hardened in the light. Thus, while there appears to be a role for carbohydrates in the cold acclimation process, the lack of parallelism between sugar content and hardiness may be interpreted as indicating 1) cold acclimation is not merely an accumulation of sugars or an osmotic effect per se, and 2) under normal conditions, the level of carbohydrates is not limiting the rate or degree of cold acclimation.     

Callus formation and plant regeneration from protonemal protoplasts of the mossAnoectangium thomsonii Mitt.

The protoplasts of the mossAnoectangium thomsonii, isolated from protonemal cells underwent divisions in 48–72 hours on modified MS medium enriched with growth regulators, 2,4-D and kinetin, 10 % sucrose and coconut water (5 %). Subculture of protoplast derived cells in a medium of relatively low osmotic potential (5 % sucrose) produced dark green calli which could be maintained completely undifferentiated.     

Rasch ablaufende Änderungen im Gehalt an löslichen Zuckern und Zellwandkohlenhydraten bei der phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.)

Summary Short term changes in the soluble sugar, starch, and cell-wall carbohydrate content of the mustard seedling have been studied in the different organs during phytochrome induced photomorphogenesis in continuous far-red. The program was: imbibition of seeds 36 hrs dark far-red irradiation. Kinetics have been followed up to 12 hrs after the onset of irradiation.There are no substantial changes in carbohydrate content in the cotyledons and the radicle. In the cotyledons in far-red after a lag-phase of 3 hrs, there is a decrease in oligosaccharide content, and after a lag-phase of 6 hrs, an increase in cell-wall synthesis. The reducing sugar and starch content is not altered upon irradiation. In the radicle immediately after the onset of far-red, there is a temporary rise in the reducing sugar and cell-wall carbohydrate content. However, 6 hrs later the values in far-red again parallel those of the dark control.The important phytochrome dependent changes take place in the hypocotyl. In far-red after a lag-phase of 3 hrs the glucose accumulation is markedly retarded, the sucrose and starch content no longer increased, and the fructose content even decreases below the 3 hrs value. The glucose: fructose ratio, which is constant in dark, is shifted in favour of glucose. The lag-phase of phytochrome controlled hypocotyl elongation is about 1 hr, the lag-phase of the inhibition of cell-wall carbohydrate synthesis is in about the same order of magnitude.There seems to be neither any immediate connection between sugar content and cell-wall carbohydrate synthesis, as shown by the difference in lag-phases, nor does there seem to be any direct relationship between hypocotyl inhibition and overall synthesis of cell-wall material. The relative inhibition of cell-wall synthesis is less than one third of that of hypocotyl elongation (Figs. 5,6). Apparently phytochrome controls hypocotyl elongation by influencing the cell-wall structure.In spite of the fact that fat degradation is higher in far-red than in dark and respiration higher in dark than in far-red (Friederich, 1968), 6 hrs after the onset of far-red the increase of total carbohydrate content declines compared with that in dark. This finding leads to the conclusion that the efficiency of the fat-carbohydrate-transformation is higher in dark than in far-red.     

Establishment and growth characteristics of a cell suspension culture of Marchantia polymorpha L. with high chlorophyll content

A green-pigmented cell suspension culture of Marchantia polymorpha was established using the medium of Murashige and Skoog with addition of organic acids of the tricarboxylic-acid cycle, vitamins and sugars plus sugar alcohols, exclusion of kinetin, and replacement of sucrose with glucose. In continuous light, the cells grew exponentially for ca. 10 days; in the dark, they grew only to a slight extent. The light-grown cells contained well-developed chloroplasts, and chlorophyll content reached almost twice that of the intact gametophyte.