Senescence in oat leaves: Changes in translatable mRNAs

Changes in translatable mRNA populations during the senescence of oat (Avena sativa L. cv. Victory) leaves were examined by analyzing the in vitro translation products of isolated RNA. Total RNA was isolated from oat leaves of 7-day-old seedlings, and also after these leaves were aged for different lengths of time under various conditions. Polypeptides from in vitro translations were separated by two-dimensional gel electrophoresis to estimate any changes in translatable mRNA populations associated with senescence. Corresponding leaf samples were monitored for loss of chlorophyll as a measure of the extent of senescence. The aging of excised leaves in the light for 4 days resulted in the disappearance or substantial quantitative decrease of a number of mRNA species, while only five new translatable mRNA species were produced. Three of these mRNAs were unique to aging of leaves under light. Two of these mRNA species were also produced during the early stages of senescence in attached leaves of seedlings grown under light. The translatable mRNA populations of leaves aged for 4 days either on intact seedlings or detached and kept in the light in the presence of kinetin were very similar. Aging of excised leaves in the dark on water for 24 h resulted in very extensive changes in translatable mRNA populations. Over thirty polypeptides disappeared or were substantially reduced in quantity, while about an equal number appeared de novo or were substantially increased in quantity. Aging of these leaves for an additional 24 or 48 h resulted in only a few additional changes in translatable mRNAs. The presence of kinetin during aging of excised leaves in the dark inhibited few of the numerous changes in mRNAs that occured during the first 24 h, but did inhibit most of the changes that occured after 48 or 72 h of aging in the dark. When leaves were first aged in the dark and then returned to light, most of the initial changes in translatable mRNAs expression were reversed. Such changes in mRNAs thus appear to be light-regulated and not necessarily associated with senescence.     

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)     

Effect of the expression of cyanamide hydratase on metabolites in cyanamide-treated soybean plants kept in the light or dark

Metabolite profiling of untransformed and cyanamide hydratase- (Cah) transformed (denoted 1C) soybean (Glycine max [L.] Merrill) leaves revealed only small differences in plants grown in the greenhouse or in the dark for 24 h, indicating that the Cah enzyme that converts cyanamide to urea has no substrates in soybean leaves and does not affect metabolism. Untransformed leaves sprayed with 0.5% cyanamide developed necrotic lesions within 2 h in the light but not in the dark. The sprayed 1C leaves showed little visible damage and accumulated high concentrations of urea, amino acids, and some sugars, but sucrose decreased over a 24 h period. The untransformed necrotic leaves also accumulated some urea and amino acids apparently due to cyanamide degradation, while sucrose and some organic acids decreased. Sprayed 1C leaves in the dark for 24 h contained very little urea and lower sugar levels. The untransformed sprayed leaves accumulated some organic acids, some sugars including sucrose, and urea and total amino acids. Unsprayed plants of both lines placed in the dark for 24 h showed increases in some amino acids and phosphate, and decreases in other amino acids, sugars, and organic acids. Thus the Cah enzyme can detoxify cyanamide by conversion to urea that is converted to amino acids. Other metabolic changes associated with leaf necrosis and darkness are also described. Principal component analysis confirmed the similarities and differences observed. Comparison of the GC-MS metabolic profiling analysis of amino acids with a dedicated system shows large differences, indicating a limitation of the former system.     

White light‐induced sugar distribution controls growth and osmotic properties in the coleoptile and the first leaf in Zea mays seedlings

The correlation of white light‐induced changes in osmotic concentration in the coleoptile and the first leaf and the growth rate of these organs in maize seedlings, was examined in relation to sugar distribution and invertase activity. One hour irradiation with white light decreased the osmotic concentration in basal zones of the coleoptile and increased it in the apical zones of the first leaf. The change in the osmotic concentration was positively correlated with the growth rate of both organs. The amount of total osmotic solutes in each zone was highly correlated with that of soluble sugars. Light decreased the activity of wall‐bound invertase in the coleoptile, but increased it in the first leaf. A high correlation existed between the content of soluble sugars and invertase activity in both organs. During 1 h incubation in the light, ca 2 µmol of soluble sugars per seedling was lost from the coleoptile and gained in the first leaf. Light promoted sugar exudation from the excised coleoptile, but the amount of soluble sugar exuded represented 5% of sugar loss from the coleoptile in intact seedlings.
These results indicate that in maize seedlings white light controls the growth rate of the coleoptile and the first leaf through the osmotic concentration. Light may have an osmoregulatory function in the control of sugar distribution between the coleoptile and the first leaf by regulating the activity of wall‐bound invertase.     

Incorporation of nitrate nitrogen in rice seedlings transferred to anaerobic conditions

Summary Incorporation of¹⁵NO₃- into amino acids was studied in 3-day-old aerobic rice seedlings (with coleoptile and root) subjected for 24h to anaerobic conditions. The incorporation of¹⁵N into glutamate, glutamine and alanine accounted for 89% and 84% of total incorporation in coleoptile and root, respectively. These findings indicate that, after the primary incorporation of¹⁵N into glutamate and glutamine, the main fate of nitrate nitrogen in rice seedlings subjected to anoxia is alanine.     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

Unusual accumulation of S-methylmethionine in aerobic-etiolated and in anoxic rice seedlings: an 1H-NMR study

An unknown signal at 2.93 ppm in 1H-NMR spectra of rice, Oryza sativa, was assigned to the methyl groups of sulphur-methylmethionine (SMM), thereby devising a new method for the determination of this compound. Rice seedlings growing aerobically in the dark and in the light engaged for the synthesis of SMM an amount of Met corresponding to 23 and 8%, respectively, of the total seed reserves of this amino acid. In etiolated shoots, SMM reached 1.2 micromol g(-1) fresh weight, an unusually high level in vegetative tissues of wild-type plants. This is compared to a value of 0.4 micromol g(-1) fresh weight in green tissues. A decreased demand for Met during growth caused the higher accumulation of SMM in etiolated, rather than green, tissues. At the same time, dark seedlings were endowed with a readily utilizable and translocable alternative form of Met, as shown by retrieval of SMM from the coleoptile. The importance of methyl group storage in SMM is shown by comparison with choline and choline phosphate pools.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Occurrence of cytochrome c-554 in a facultative methylotroph,Protaminobacter ruber strain NR-1, during bacteriochlorophyll formation

A facultative methylotroph, Protaminobacter ruber was grown under two different conditions (aerobically grown under light, and aerobically in the dark after a light period). Bacteriochlorophyll was synthesized inducibly in the cells which were initially grown in the ligt and then grown in the dark, while bacteriochlorophyll was not found in the cells cultured under continuous light. Cytochrome c-554 was solely synthesized parallel to bacteriochlorophyll after switching from light to dark conditions. Both cytochrome c-554 and bacteriochlorophyll levels in the membrane preparation reached to a plateau in 24 h after switching from light and dark conditions. This cytochrome was membrane-bound and its M _r was 45,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis. The midpoint potential was 358 mV at pH 7. Other major membrane-bound cytochromes and two soluble cytochromes were present in both types of cells and their content did not change irrespective of growth conditions.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Bchl bacteriochlorophyll     

The exudation of solutes during senescence of oat leaves

Exudation of cations and of amino acids from detached oat seedling leaves ( Avena sativa L. cv. Victory) floated on water or solutions was measured under varied conditions. A small amount of exudation in the first 4 h, greater in the dark than in white light, is followed, one to 8 days later, by a relatively great increase which approximately accompanies senescence. This second increase is delayed by kinetin and is accelerated by abscisic acid or methyl jasmonate. both of which accelerate senescence. A nitrogen atmosphere (in darkness) causes immediate large exudation, mainly of potassium ions, and this is effectively delayed by light. There are two exceptions to the parallel between exudation and senescence; n-pentanol, which strongly delays senescence in darkness, nevertheless increases exudation both of potassium and of amino acids, probably due to a direct effect on the plasmalemma. Cycloheximide, which also delays senescence, increases the exudation somewhat. Kinetin prevents or delays exudation under all conditions. Thus, the permeability of the plasmalemma increases greatly along with other criteria of senescence, but this change is probably not the principal cause of the senescence syndrome. Some of the effects are considered to result from reduction in available energy and others from a direct influence on plasmalemma permeability.     

Activity and characterization of mixed organic compounds extracted from Rhodobacter sphaeroides as alternative materials to serum for mammalian cell growth

Photosynthetic microorganisms produce relatively large amounts of physiologically active materials which stimulate the physiological activity of other organisms. In this study, mammalian HeLa cells were cultured in different culture media which were Dulbecco's modified Eagle medium (DMEM) with newborn calf serum (NCS), and DMEM including different types of physiologically activating compounds (PACs) extracted from Rhodobacter sphaeroides grown under various culture conditions. R. sphaeroides was grown under the following five different culture conditions: anaerobically in the light, anaerobically in the dark and treated with dimethyl sulfoxide, aerobically in the dark for 48 h, in the light for 48 h, and in the light for 24 h and changed after previous culturing in the dark for 24 h. The growth of HeLa cell was measured by cell counting using a hemocytometer, and the fluorescent intensities of cellular lysosomes were measured to check the level of cellular stress caused by adding PACs. The growth of HeLa cells cultured in DMEM with PACs extracted from R. sphaeroides aerobically grown under dark conditions was enhanced compared to that of cells grown with NCS. We also found that a high concentration of pigments such as bacteriochlorophylls and carotenoids and a high concentration of arginine produced by R. sphaeroides aerobically grown in the dark were implicated in increased growth of the HeLa cells. Therefore, our results suggest that PACs extracted from R. sphaeroides aerobically cultured in dark conditions can enhance the physiological activity of mammalian cells and serve as nontoxic and bioavailable materials.     

Photosynthetic Development of Anaerobically Grown Rice (Oryza sativa) after Exposure to Air

During anaerobic germination, rice produces a coleoptile devoid of carotenoid and chlorophyll. Further development and greening of the shoot occur upon exposure of the seedlings to air. In this study, a comparison was made between anaerobically (N₂) germinated rice, greened upon exposure to air, and air/dark (A/D) germinated seedlings, greened upon exposure to light. After exposure to air, N₂-grown seedlings had a 76-hour lag before net oxygen evolution occurred compared to a 6-hour lag for A/D-grown seedlings. After 98 h of greening, N₂-grown seedlings reached a rate of oxygen evolution equivalent to that of A/D-grown seedlings after 24 hours. Chlorophyll and carotenoid content showed a similar lag, but did not reach the level found in A/D-grown seedlings even after 124 hours of exposure to air. RuBPcase activity also lagged in N₂-grown seedlings, ultimately reaching greater values than in the `greened' A/D-grown seedlings. Phosphoenolpyruvate carboxylase activity was constant and low in all treatments except for a transient increase after 24 hours of greening of the N₂-grown seedlings.     

Amino acid composition of Phaseolus aureus L. seeds and seedlings

The amino acid composition of seeds and 10-day seedlings of mung bean (Phaseolus aureus L.) as well as that of proteins of Ph. aureus seedlings was measured. The seedlings were grown under different conditions of nitrogen nutrition in the light and in the dark. Ph. aureus seeds showed a high content of some essential amino acids. As compared with seeds, the seedlings had higher concentrations of aspartic acid and isoleucine and lower concentrations of glutamic acid, lysine and histidine. Proteins of Ph. aureus seedlings showed greater amounts of isoleucine, phenylalanine, threonine, lysine and lower quantities of glutamic acid. Methionine and cystine were limiting amino acids.     

The Relationship between Sugar and Amino Acid Export to the Phloem in Young Wheat Plants

The relationship between amino acid and sugar export to thephloem was studied in young wheat plants (Triticum aestivumL. ‘Pro-INTA, Isla Verde’) using the EDTA-phloemcollection technique. Plants grown with a 16 h photoperiod showeda rapid decrease in the concentration of sugars and amino acidsin the phloem exudate from the beginning of the dark period.When plants grown with a 16 h photoperiod were kept in the darkfor longer than 8 h the free amino acid content in leaves andexudate (on a dry weight basis) increased continually throughoutthe 72 h of darkness. During the first 24 h of darkness thesugars in the phloem exudate decreased to 30% of the initialvalue, and returned to the control level when plants were returnedto light. When plants grown under low light intensity for 10d were transferred to high light intensity, they showed an increasein leaf sugar content (dry weight basis) after 3 d but therewere no differences in leaf free amino acid content (dry weightbasis) compared to low-light plants. The sugar concentrationin the phloem exudate was increased by higher light intensities,but there was no difference in the amino acid concentrationof the phloem exudate, and thus the amino acid:sugar ratio inthe phloem decreased in the high-light plants. The present resultssuggest that amino acids can be exported to the phloem independentlyof the export of sugars. Copyright 1999 Annals of Botany Company Sugar exudation, amino acid transport, nitrogen, phloem, transport, wheat, Triticum aestivum L.     

Physiological and ultrastructural changes in Stellaria media following treatment with fluroxypyr

The physiological and ultrastructural changes induced by fluroxypyr (4-amino-3, 5-dichloro–6–fluoro–2–pyridyloxyacetic acid) are investigated in a susceptible weed species, Stellaria media. The sequence of symptoms was an initial petiole curvature followed by stem elongation and thickening prior to leaf senescence, stem and petiole necrosis and plant death. Light and electron microscopy revealed leaf tissue containing cells with disrupted and swollen chloroplasts and a disrupted tonoplast. Within the stem tissue there was extensive meristematic differentiation and adventitious root development prior to stem tissue necrosis. Increased levels of reducing sugars and amino acids in treated foliar tissue indicated reserve mobilisation during the initial stages of symptom development. Fluroxypyr induced oat coleoptile elongation with an optimum concentration at 5 × 10^-5 m compared to 10^-4 m for auxin (IAA). A S. media shoot explant system revealed similar elongation which was maximal during the first 24 h of treatment. Uptake and incorporation of ¹⁴C-leucine was stimulated by 96 h after treatment. It is concluded that fluroxypyr activity against S. media is characterised by an initial cell elongation and reserve mobilisation, followed by extensive cell proliferation, increased RNA and protein synthesis, and ultimately plant death.     

Compartmentation of labeled fixation products in intact mesophyll protoplasts from Avena sativa L. after in-situ inhibition of the chloroplast phosphate translocator

Leaf mesophyll protoplasts of oat (Avena sativa L.) were allowed to fix ¹⁴C-labeled bicarbonate in the absence or presence of pyridoxal phosphate (PLP), a specific inhibitor of the phosphate translocator of the inner envelope membrane of chloroplasts. The incubation was terminated by a method of rapid integrated protoplast homogenization and fractionation, and compartmented levels of label contained in sugars, phosphate esters, amino acids and organic acids were determined. The results show that the addition of PLP to a suspension of intact protoplasts causes an accumulation of phosphate esters in the chloroplasts stroma for up to 2.5 min of incubation, with a corresponding decrease in the cytosol. Prolonged treatment of protoplasts with PLP in the light resulted in a decrease of starch-associated label, combined with higher levels of labeled sugars in the cytosol, indicating a switch from phosphorolytic to hydrolytic starch degradation. Together with the determination of pool sizes of triose phosphates and of inorganic phosphate, the results demonstrate that the method employed is an important tool in investigating processes of intracellular regulation. They are discussed with respect to the permeability and possible side reactions of PLP, as well as in the light of reports on PLP action on isolated chloroplasts.Abbreviations P_i orthophosphate - PLP pyridoxal 5-phosphate - TP triosephosphate     

Root growth and gravireaction: Influence of the shoot

The kinetics of root gravicurvature of intact maize ( Zea mays L., cv. LG 11) seedlings can be separated into two steps. From 0 to 2 h, a rapid downward bending, enhanced by light, occurred. During the next 4 h (2 to 6 h) no further curvature appeared in the dark, whereas it continued in light. The final root gravicurvature was greater for light-than dark-treated seedlings. Growth was particulary inhibited during the first 2 h.
When intact seedlings were placed horizontally and returned to the dark after a 4 h light pretreatment in a vertical position, only the second step (2 to 6 h) was changed. A negative gravitropism, associated with a stimulation of growth, appeared. Thus, when gravireaction took place in darkness, the final root gravicurvature was similar for the light-pretreated intact seedlings and the dark control. Using apical root segments, this phase of negative gravicurvature was not observed although the stimulation of gravicurvature of light-pretreated roots remained. Similar experiments were performed with seedlings having coleoptile or endosperm removed, after intact seedlings had been exposed to a light pretreatment, and gravireaction took place in the dark. Results indicated that these two organs could play an essential role in the development of gravicurvature and in the regulation of the light effects, respectively.     

Photoactivation of Chlorophyll Synthesis and Cytochrome Oxidase Activity in Anaerobically Germinated Seedlings of Echinochloa crusgalli var. Oryzicola

When seeds of Echinochloa crusgalli var. oryzicola are germinated in dark anaerobic conditions (99.995% N₂), the seedlings do not have detectable protochlorophyll(ide). Two hours after exposure to light aerobic conditions, they begin to synthesize chlorophyll. The lag in greening is shorter in seedlings exposed to light for 24 hours before exposure to air. Seedlings maintained in light anaerobic conditions exhibit no lag in greening upon transfer to an aerobic environment. Preillumination of anaerobically grown seedlings does not result in any chlorophyll accumulation. Phytochrome is probably the receptor for photoactivation of chlorophyll synthesis, since activation is achieved by red light alone, but not by far red light or red plus far red light. The cytochrome oxidase activity in anaerobically germinated seedlings is 30% of the normal level found in aerobically grown seedlings. Preillumination was also found to activate the ability of anaerobically germinated seedlings to increase their cytochrome oxidase activity upon exposure to air.     

Establishment of far‐red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene

A transgenic wheat line over‐expressing an oat phytochrome A gene under the control of the constitutive maize ubiquitin promoter was generated using a biolistic particle delivery system from immature wheat embryos. The resulting line showed increased levels of total phytochrome A protein in both dark‐grown and light‐grown plants. When grown under continuous far‐red light, seedlings of this line showed additional inhibition of the coleoptile extension in comparison with wild‐type seedlings. Unlike the response of wild‐type seedlings to continuous far‐red, this additional inhibition was dependent on fluence rate and was not observed under half‐hourly pulses of far‐red delivering the same total fluence as the continuous irradiation treatment. These observations suggest that increase in phytochrome A levels in wheat leads to the establishment of a far‐red high irradiation reaction in this monocotyledonous plant. Exposure to continuous red light caused a similar inhibition of coleoptile extension in both the wild types and the transgenic seedlings. When wild‐type seedlings were grown under continuous far‐red, their coleoptiles remained completely colourless and first leaves remained tightly rolled. In contrast, transgenic seedlings grown in the same conditions produced significant levels of anthocyanins in their coleoptiles and their first leaves became unrolled. Taken together, our data suggest that the increased levels of phytochrome A in wheat can change the type of response of some developmental processes to light signals, leading to the generation of a high irradiance reaction which is otherwise absent in the wild types under the conditions used.     

Induction and Turnover of Nitrate Reductase in Zea mays (Influence of Light)

Both light and NO3- are necessary for the appearance of nitrate reductase (NR) activity (NRA) in photosynthetic tissues. To define the light effect more precisely, we examined the response to light/dark transitions on NRA, NR protein (NRP), and NR mRNA in 6-d-old maize (Zea mays cv W64A x W182E) seedlings that had been grown in a light/dark regime for 5 d and then induced with 5 mM KNO3 for 24 h. The decay of NRA and NR mRNA in the shoot was immediate, but there were only minor changes in NRP during the initial 4 h in the dark. In root tissues, in contrast, there was a 4-h delay in the loss of NRA, NRP, and NR mRNA after transfer to the dark. When the seedlings were returned to light after a 2-h interval in the dark, shoot NRA reached 92% of the initial levels within 30 min of illumination. These results indicate that in the shoots (a) NR message production requires light and (b) the NRP that appears with light treatment and that is active is inactivated in the dark. The NRP can be reactivated when the light is turned on after short periods of darkness (2 h). Root tissues, on the other hand, probably respond to the supply of photosynthetically produced metabolites rather than to immediate products of the light reactions of photosynthesis.