Organ‐specific localization and molecular properties of three soluble invertases from Lilium longiflorum flower buds

Three soluble invertase (EC 3.2.1.26) isoforms from Easter lily ( Lilium longiflorum Thunb. cv. Nellie White) flower buds were purified to apparent homogeneity. Non‐denaturing PAGE showed one band for all three invertases that corresponded to the invertase activity. SDS‐PAGE of purified invertase I gave a single band at 78 kDa, whereas invertases II and III gave three bands at 54, 52 and 24 kDa. Antibodies against tomato fruit acid invertase and Urtica dioica leaf acid invertase recognized all three invertase isoforms, whereas antibodies against wheat coleoptile acid invertase recognized only 56‐ and 54‐kDa bands of invertases II and III. Antibodies against wheat coleoptile invertase recognized the 54‐ and 52‐kDa proteins from crude extracts of all flower organs, and a 72‐kDa protein in both leaf and bulb scale extracts. All three invertases bound to Con‐A peroxidase. Deglycosylation of invertase I with glycopeptidase F was complete and resulted in a peptide of 75 kDa. Invertases II and III were deglycosylated partially by glycopeptidase F and resulted in proteins of 53, 51, 50 and 22 kDa. Invertase I was localized only in anther and filament, whereas the other two isoforms were present in all flower organs.     

Changes in cellular osmotic potential and mechanical properties of cell walls during light-induced inhibition of cell elongation in maize coleoptiles

The growth rate of maize ( Zea mays L. cv. Cross Bantam T51) coleoptiles in the dark was highest at the basal zone and decreased towards the tip. Growth was strongly inhibited by white fluorescent light (5 W m⁻²), especially in the basal zone of coleoptiles. Light irradiation caused an increase in the values of stress-relaxation parameters, the minimum stress-relaxation time and the relaxation rate and a decrease in the extensibility (strain/stress) of the cell walls at all zones. In addition, during growth, the accumulation of osmotic solutes was strongly inhibited by white light irradiation, resulting in an increased osmotic potential. The influences of white light on the mechanical properties of the cell wall and the osmotic potential of the tissue sap were most prominent in the basal zone. Significant correlations were observed between the increment of coleoptile length and the mechanical properties of the cell walls or the osmotic potential of the tissue sap and osmotic solutes content. Furthermore, light inhibited the outward bending of split coleoptile segments. These facts suggest that white light inhibits elongation of maize coleoptiles by modifying both the mechanical properties of the cell walls and cellular osmotic potential, which control the rate of water uptake.     

A unique pantoyllactone glycoside system is activated in rice seedlings developing aerobically in the dark

Rice ( Oryza sativa L.) seedlings developing aerobically in the dark accumulated in the expanding coleoptile millimolar amounts of pantoyllactone glucoside (PLG) together with sugars and amino acids. Following leaf emergence and seedling development, the transformation of PLG into pantoyllactone primeveroside (PLP) was initiated inside the coleoptile, whose senescence was suspended in spite of the exhaustion of seed reserves and cessation of shoot growth. PLG, PLP and most of the sugars and amino acids remained inside the coleoptile. Light administration triggered shoot greening and coleoptile senescence that was accompanied by the conversion of PLG into the shoot-translocatable species PLP and by the utilization of the coleoptile-stored metabolites. Light-induced retrieval of amino acids and sugars from the coleoptile was 90% complete within 24 h while translocation of pantoyllactone glycosides started only 24–48 h after light administration. Conversely, no accumulation of PLG and PLP was detected in seedlings germinated and grown either aerobically in the light or in the dark under hypoxic conditions. The possible significance of the activation of the PLG-PLP system under specific environmental conditions is discussed.     

Changes in the pool of soluble sugars induced by dehydration at the heterotrophic phase of growth of wheat seedlings

Loss of dehydration tolerance coincides with a shift from heterotrophy to autotrophy during post-germination growth of spring wheat seedlings. This critical stage falls on the fifth day following imbibition. Till the sixth day of experiment light had no effect on dry weight of the seedlings but the survival of six day old seedlings was reduced by half upon dehydration. Germinating seeds in the presence of 5 mM glucose, fructose, mannose or sucrose did not promote seedling growth but either increase (glucose, fructose) or decreased (mannose, sucrose) the survival of dehydrated seedlings. Protection against dehydration by the former sugars was correlated, irrespective of the seedling age, with the decrease of sugar pool in seeds and increase in shoots (coleoptile and first leaf) and roots. The opposite changes were provoked by the sugars hampering seedling survival. Generally, survival of wheat seedlings was not correlated with the size of soluble sugar pool but its distribution and composition. Lower mobilisation of soluble sugars in seed, lower proportion of reduced sugars to sucrose and higher share of raffinose is characteristic for the tolerant four day old seedlings and those grown in the media containing glucose or fructose. The results presented indicate that higher proportion of reduced sugars to sucrose and lower share of raffinose in six day old seedlings seems to be associated with the loss of dehydration tolerance of these seedlings, despite heterotrophic character of growth.     

Gibberellin-enhanced sugar accumulation in growing subhooks of etiolated Pisum sativum seedlings. Effects of gibberellic acid, indoleacetic acid and cycloheximide on invertase activity, sugar accumulation and growth

The possible involvement of invertase in the action of gibberellic acid (GA) on stimulating sugar accumulation in growing subhooks of Alaska pea ( Pisum sativum L. cv. Alaska) was studied. GA and indoleacetic acid (IAA) stimulated elongation growth to a similar extent. GA, in contrast to IAA, increased the amount of soluble sugars in the subhook. GA substantially increased invertase activity whereas IAA did not. These results suggest that the mode of action of GA and IAA differs, although both stimulate pea subhook growth.
Cycloheximide (CH) inhibited the effect of GA on invertase activity, accumulation of soluble sugars, and elongation growth. Good correlations were found between invertase activity, the amount of soluble sugars and growth. The results suggest that GA-induced enhancement of sugar accumulation in the subhook cells is dependent on increased invertase activity. The sugar accumulated in the subhook may be involved in growth promotion by GA.     

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m^-2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine-and tyrosine-ammonia-lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize ( Zea mays L. cv. Cross Bantam T51). Growth rate of dark-grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p -coumaric acid ( p -CA) increased by severalfold in cell walls of light-grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p -CA and the decrease in cell wall extensibility. Light decreased the wall-bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p -CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.     

Osmotic relations during elongation growth in hypocotyls of Helianthus annum L.

The relationship between growth, change in cell osmotic pressure and accumulation of osmotic solutes was investigated in hypocotyls of sunflower (Helianthus annum L.) seedlings. During growth in darkness the osmotic pressure decreased by 50% between days 2 and 6 after sowing. After irradiation of dark-grown seedlings with continuous white light (WL) an inhibition of hypocotyl growth was measured, but the osmotic pressure of the growing cells was not lower than in the dark-grown control. Growth in darkness and after WL irradiation was accompanied by an increase in the amount of osmotic substances (soluble sugars) which was proportional to the increase in length of the organ. During growth in continuous WL the cell osmotic pressure decreased by 45 % between days 2 and 6 after sowing. The transfer of WL-grown seedlings to darkness (“re-etiolation”) resulted in a rapid acceleration of hypocotyl growth, but the cell osmotic pressure was the same as that of the WL grown control. Growth in continuous WL was accompanied by a corresponding accumulation of osmotic substances (soluble sugars). The transition from WL to darkness resulted in an enhanced accumulation of osmotica and an increase in cell-wall extensibility. The results indicate that the relative maintenance of cell osmotic pressure during rapid hypocotyl growth in darkness is caused by an enhanced accumulation of soluble sugars into the growing cells of the organ.     

Pigment Accumulation and Photosynthesis in Developing Rye Coleoptiles

Rye seedlings (Secale cereale L.) were grown in darkness or irradiated with white light (WL) of medium intensity (100 μmol quanta . m^?2 . s^?1). In the coleoptile and primary leaf of irradiated plants synthesis of chlorophylls a and b with average ratios of 2.4 and 2.7, respectively, were measured. The chlorophyll content of the fully green primary leaf was about 10-fold larger than that of the pale green coleoptile. In darkness a large increase in carotenoid content occurred in the primary leaf, but in the coleoptile the level of carotenoids remained very low. In both organs a WL-induced increase in carotenoid synthesis was observed. The level of anthocyanin was likewise enhanced by WL. In the coleoptile and primary leaf of irradiated seedlings light-dependent oxygen evolution was measured. The specific activity of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase was enhanced after WL treatment. The results indicate that the mature rye coleoptile is a photosynthetically active plant organ.     

The role of amino acids and sugars in supporting of osmotic homeostasis in maize seedlings under salinization conditions and treatment with synthetic growth regulators

Stress state in plants caused by salinization conditions is characterized by the disturbance of ionic and osmotic homeostasis. The maintenance of the latter is reached by accumulation of osmolytes including free amino acids and soluble sugars in cells. The free amino acid level in the 8-day-old control seedling leaves was higher, than in the roots, whereas the contrary picture was observed in 17-day-old plant tissues. At the same time 8-day-old seedling roots contained more total sugars, than leaves, although the reduced sugar content was nearly a half of the total sugar content. A decrease of both total and reduced sugar levels was observed in 17-day-old seedling tissues. One-day exposure of 7-day-old seedlings to 0.1 M NaCl increased the free amino acid content especially in roots, than in leaves, and the total sugar content in maize leaves, whereas in roots this level remained without changes. The prolongation of salt exposure to 10 days leads to osmolyte content decrease. The seed treatment with Methyure and Ivine intensified accumulation of free amino acids and soluble sugars in the root and leaf tissues under salinization conditions.     

Excess nickel modulates activities of carbohydrate metabolizing enzymes and induces accumulation of sugars by upregulating acid invertase and sucrose synthase in rice seedlings

The effects of increasing concentrations of nickel sulfate, NiSO₄ (200 and 400 μM) in the growth medium on the content of starch and sugars and activity levels of enzymes involved in starch and sugar metabolism were examined in seedlings of the two Indica rice cvs. Malviya-36 and Pant-12. During a 5–20 day growth period of seedlings in sand cultures, with Ni treatment, no definite pattern of alteration in starch level could be observed in the seedlings. In both roots and shoots of the seedlings Ni treatment led to a significant decrease in activities of starch degrading enzymes α-amylase, β-amylase, whereas starch phosphorylase activity increased. The contents of reducing, non-reducing, and total sugars increased in Ni-treated rice seedlings with a concomitant increase in the activities of sucrose degrading enzymes acid invertase and sucrose synthase. However, the activity of sucrose synthesizing enzyme sucrose phosphate synthase declined. These results suggest that Ni toxicity in rice seedlings causes marked perturbation in metabolism of carbohydrates leading to increased accumulation of soluble sugars. Such perturbation could serve as a limiting factor for growth of rice seedlings in Ni polluted environments and accumulating soluble sugars could serve as compatible solutes in the cells under Ni toxicity conditions.     

Effects of Short-term Red Radiation and Choline Compounds on Cytokinin Content, Chlorophyll Accumulation and Photomorphogenesis in Wheat Seedlings

Effects of choline compounds (2-chloroethyltrimethylammonium chloride and 2-ethyltrimethylammonium chloride) as well as red radiation (R) pulse on the dynamics of cytokinin changes, growth and chlorophyll (a + b) accumulation were studied during the growth and greening of etiolated wheat seedlings (Triticum aestivum L., var. Mironovskaya-808). The seedlings were grown for 120 h in the dark and then exposed for 72 h to white light. Pre-treatment of caryopses with cholines and pre-irradiation of etiolated seedlings with R inhibited elongation of both coleoptile and first leaf; but the same factors accelerated these growth responses when seedlings were exposed to white light. Chlorophyll (Chl) accumulation and the first leaf appearance from coleoptile were accelerated by the pre-treatments as well. Far-red radiation (FR) reversed all effects of R but choline pre-treatment eliminated partly R/FR photoreversibility. Two compounds with high cytokinin activity (tested on a fresh weight basis by the bioassay with Amaranthus caudatus L.) were found in shoots and first leaves. One of them had R_f, UV absorbance spectrum and the biological activity similar to N⁶-(Δ²-isopentenyl)adenosine. Another cytokinin-like substance was not identified with the used standards. Stimulation of greening by R pulse and cholines was accompanied with accelerated accumulation of both cytokinin-like substances. We conclude that the influence of R and cholines on the concentration of substances with cytokinin activities detected in the leaves might be involved in the stimulation of Chl accumulation.     

Inhibition of the Coleoptile Growth in Avena sativa by Endosperm Removal--Changes in Auxin, Osmotica and Cell Wall

Removal of the endosperm from 84-h-old etiolated oat seedlingsstrongly retarded the subsequent growth of coleoptiles. Thecontribution of the endosperm to coleoptile growth was studied.Endosperm removal was found to: (1) decrease the endogenouslevel of indole-3-acetic acid (IAA) in the coleoptile tip. IAAapplied to the coleoptile tip stimulated coleoptile growth inseedlings with and without the endosperm. The sensitivity ofthe coleoptile to a suboptimal concentration of IAA was higherin seedlings without the endosperm than in intact ones. At theoptimal concentration of IAA, however, the final length of thecoleoptile was larger in intact seedlings than in those withoutthe endosperm. (2) decrease the concentration of the solublesugars and amino acids in the cell sap. (3) retard the increasein the amount of polysaccharides in the cell wall of the coleoptile,particularly noncellulosic ones. (4) make the cell wall mechanicallyrigid according to stress-relaxation analysis of the cell wall.(5) induce an increase in the osmotic potential of the coleoptilecell sap. From these results, it was concluded that the endosperm suppliesthe coleoptile with IAA, sugars and amino acids, thus promotingcoleoptile growth. (Received September 24, 1987; Accepted February 3, 1988)     

Effects of chilling stress on the accumulation of soluble sugars and their key enzymes in <Emphasis Type="Italic">Jatropha curcas</Emphasis> seedlings

As osmolytes and signaling molecules, soluble sugars participate in the response and adaptation of plants to environmental stresses. In the present study, we measured the effect of chilling (12 °C) stress on the contents of eight soluble sugars in the leaves, cotyledons, stems, and roots of Jatropha curcas seedlings, as well as on the activities of eight rate-limiting enzymes that are critical to the metabolism of those soluble sugars. Chilling stress promoted both starch hydrolysis and soluble sugar accumulation. The soluble sugar contents of the leaves and cotyledons were affected more than that of the stems and roots. Meanwhile, the activities of the corresponding metabolic enzymes (e.g., β-amylase, uridine diphosphate glucose phosphorylase, and sucrose phosphate synthase) also increased in some organs. The gradual increase of soluble neutral alkaline invertase activity in the four studied organs suggested that sucrose catabolic production, such as glucose and fructose, was especially important in determining resistance to chilling stress and hexose signal transduction pathway. In addition, the substantial accumulation of raffinose family oligosaccharides and increase in corresponding metabolic enzyme activity suggested that galactinol and raffinose play an important role in determining the chilling resistance of J. curcas. Together, these findings establish a foundation for determining the relationship between the chilling resistance and soluble sugar accumulation of J. curcas and for investigating the mechanisms underlying sugar signaling transduction and stress responses.     

Growth rate,photosynthetic activity,and leaf development of Brazil pine seedlings (Araucaria angustifolia[Bert.] O. Ktze.)

Seedlings of Brazil pine, a large-seeded South American conifer, were grown in a climate chamber to investigate vertical growth pattern and the time course of leaf development. We examined shoot growth, photosynthetic performance and markers of leaf maturation such as contents of soluble sugars and activities of sucrose-phosphate synthase (SPS), neutral invertase (nI) and sucrose synthase (Susy). The daily increment of shoot length showed an optimum curve during the first 70 days after germination. The low growth rate during the first 20 days of development correlated with net CO² emission of the seedling. Analyses of leaf maturation markers in older seedlings revealed low sucrose/hexose ratios and high activities of nI and Susy in the uppermost leaves. Although the SPS/Susy ratio was low in these leaves the extractable SPS activity did not change significantly among leaves of different age. The photosynthetic light compensation points of young leaves were about 2-fold higher than those of mature leaves and their photosynthetic capacity was only 50% as high. Our results indicate that a rapid maturation of leaves of Brazil pine seedlings may reduce the respiratory loss of carbohydrates and that the mobilisation of seed storage compounds supports initial shoot growth under light-limiting conditions which may occur in the forest-grassland succession zone.     

Changes in the Autolytic Activities of Maize Coleoptile Cell Walls during Coleoptile Growth

Autolytic activities of coleoptile cell walls were measuredin developing maize seedlings. The major neutral sugar componentsof the cell wall polysaccharides were arabinose, xylose andglucose. The quantities of all these components per coleoptileincreased for 5 d after germination, suggesting that levelsare augmented by biosynthetic processes during coleoptile growth.However, cell wall preparations isolated from the coleoptilesalso revealed increasing rates of autolytic activity directedtoward each of the sugar components. This result suggests thatthe constitutive hydrolytic activities expressed by cell wallsalso increase as a function of coleoptile age. The proportionof glucose in autolysis products relative to that present inthe cell walls specifically increased with coleoptile age, whilethe ratios for arabinose and xylose decreased. Kinetic analysesof autolysis demonstrated that the reactions specific for pentosesat the early growth stage are transient events and that initiallow rates of glucan autolysis increased sharply and persistedlonger. In these experiments the products of glucan autolysiswere largely monomeric while those of the pentose-specific reactionsconsisted of both monomeric and polymeric sugars. Based on theseresults, we concluded that two distinct phases of autolyticactivities are expressed in the mediation of cell wall polysaccharidemetabolism in situ. (Received July 17, 1996; Accepted November 25, 1996)     

Water stress and light intensity effects on growth and nocturnal acid accumulation in a terrestrial CAM bromeliad (Bromelia humilis Jacq.) under natural conditions

Summary Seasonal variations in CAM performance of sunexposed and partially shaded populations of Bromelia humilis were measured under natural conditions in a semi-arid region in northern Venezuela. The sun population consisted of smaller plants, with lower chlorophyll and total nitrogen contents per unit leaf area compared with plants from the partial-shade population. During the dry season CAM activity, assessed as nocturnal acid accumulation, was higher in the partial-shade population. Acid accumulation was stimulated by irrigation in both populations within 24 h after treatment. Daily changes in concentration of soluble sugars were opposite to leaf acidity indicating their role as carbon source for acid synthesis during the night. The change in nocturnal sugar concentration was always more than the amount required for acid accumulation, suggesting other carbohydrate-consuming processes such as transportation of sugars out of the leaf. CAM activity was higher during the rainy season, and differences between populations were smaller. At the end of the rainy season reduction of CAM activity caused by drought was first detected in the sun population. Measured ratios of glucan/soluble sugar show a higher proportion of readily utilizable sugars during periods of active CAM and growth. Under conditions of continuous high light intensity and air temperature leading to all year round high potential evaporation in semiarid tropical regions, fully exposed populations of B. humilis show a pronounced reduction of metabolic activity. Partial shade favours growth and CAM activity in this constitutive CAM species. It is concluded that water stress, and not light intensity, is the predominant limiting factor for growth of this species under natural conditions.     

质膜H~＋－ATP酶在玉米叶片渗透调节中的作用

干早胁迫下,玉米幼叶生长部位质膜Ｈ＋－ＡＴＰ酶活性显著上升,游离脯氨酸、可溶性糖和无机离子亦同时在玉米叶片生长部位大量积累,二者之间呈明显的正相关．质膜Ｈ＋－ＡＴＰ酶的专一性抑制剂Ｎａ３ＶＯ４在干旱胁迫下强烈抑制游离脯氨酸的积累,说明ＰＭＨ＋－ＡＴＰａｓｅ参与了玉米叶片的渗透调节过程．     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Hydrogen peroxide pretreatment induces osmotic stress tolerance by influencing osmolyte and abscisic acid levels in maize leaves

Hydrogen peroxide (H₂O₂) functions as a signal molecule in plants under abiotic and biotic stresses. Leaves of detached maize (Zea mays L.) seedlings were used to study the function of H₂O₂ pretreatment in osmotic stress resistance. Low H₂O₂ concentration (10 mM) which did not cause a visual symptom of water deficit (leaf rolling) was applied to the seedlings. Exogenous H₂O₂ alone increased leaf water potential, endogenous H₂O₂ content, abscisic acid (ABA) concentration, and metabolite levels including soluble sugars, proline, and polyamines while it decreased lipid peroxidation and stomatal conductance. Osmotic stress induced by polyethylene glycol (PEG 6000) decreased leaf water potential and stomatal conductance but enhanced lipid peroxidation, endogenous H₂O₂ content, the metabolite levels, and ABA content. H₂O₂ pretreatment also induced the metabolite accumulation and improved water status, stomatal conductance, lipid peroxidation, ABA, and H₂O₂ levels under osmotic stress. These results indicated that H₂O₂ pretreatment may alleviate water loss and induce osmotic stress resistance by increasing the levels of soluble sugars, proline, and polyamines thus ABA and H₂O₂ production slightly decrease in maize seedlings under osmotic stress.