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.     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^?1 h^?1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L^?1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L^?1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Change in Gene Expression in Radish Cotyledons during Dark-Induced Senescence

Cotyledons detached from light-grown radish (Raphanus sativusL. cv. Comet) seedlings were used as a model system to studythe changes in nuclear gene expression during dark-induced senescenceof green leaves. Polyadenylated RNA was prepared from the cotyledonsat different times and then translated in a wheat germ system.Approximately 1,000 different polypeptides of the translationproducts were separated from each other by two-dimensional gelelectrophoresis. As judged from the density of autoradiographicspots of the translation products, the induction of senescenceby dark treatment involved an increase in 26 species, a decreasein 11 species, and a temporary increase and subsequent decreasein 8 species of translatable mRNA. A similar pattern of changein protein synthesis was also observed in the dark-treated cotyledonswhen the cotyledons were pulse-labeled with ³⁵S-methionine andthe soluble proteins separated by two-dimensional gel electrophoresis,though the polypeptide pattern on the gel did not coincide exactlywith those of the cell-free translation products. These findingsstrongly suggest that the process of leaf senescence is notsimply a passive and gradual death of the tissue, but involvesa drastic and sequential response of the cells to environmentalstimuli with respect to the gene expression of the cells. (Received July 21, 1987; Accepted September 30, 1987)     

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.     

Synthesis of Translatable mRNA for Phytochrome during Imbibition in Embryonic Axes of Pisum sativum L.

The activity of translatable mRNA for phytochrome was measuredin excised embryonic axes of Pisum sativum L. during imbibitionboth in the dark and under continuous irradiation with whitelight. When measured in cell-free protein synthesis systemsof both rabbit reticulocyte lysate and wheat germ extract, theactivity of translatable mRNA for phytochrome was not detectedin dry quiescent axes but increased rapidly after imbibitionin the dark. After 24 h imbibition, the level of translatablemRNA for phytochrome, in terms of the incorporation of [³⁵S]methioninein the wheat germ system, was ca. 0.0034% of total translatablemRNA. In the presence of 0.5 µg ml^–1 -amanitin,the appearance of translatable mRNA for phytochrome was inhibitedby 60%, while 2 µg ml^–1 -amanitin was almost completelyinhibitory. This indicates that the synthesis of translatablemRNA for phytochrome in embryonic axes begins upon imbibition. When the axes were imbibed under continuous white light, theactivity of phytochrome mRNA increased as rapidly during thefirst 3 h as in the dark. After this time, the activity wasmarkedly lower than in the dark. Nevertheless, during the 24h of imbibition, activity in the light was always found to bemore than half of that in the dark. These results indicate thatin germinating pea axes the level of translatable mRNA for phytochromeis partially repressed by light. (Received June 5, 1985; Accepted September 2, 1985)     

Studies on the Photoreceptors for the Promotion and Inhibition of Flowering in Dark-Grown Seedlings of Pharbitis nil Choisy

Three-day-old etiolated seedlings of Pharbitis nil were exposedto red light for 10 min and sprayed with N⁶-benzyladenine beforetransfer to a 48-h inductive dark period, after which they weregrown under continuous white light. A second red irradiationpromoted flowering when given at the 5 and 24th hour of theinductive dark period but inhibited flowering at the 10 and15th hour. Far-red light inhibited flowering when given at anytime during the first 24 h of the dark period. Red/far-red reversibilitywas clearly observed at the 0, 5, 10 and 24th hour, but notat the 15th hour when both red and far-red lights completelyinhibited flowering. The action spectrum for the inhibition of flowering at the 15thhour of the inductive dark period had a sharply defined peakat 660 nm and closely resembled the absorption spectrum of theP_R form of phytochrome. The photoreceptors involved in thesephotoreactions are discussed. (Received June 10, 1983; Accepted July 6, 1983)     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^–1 h^–1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1l L^–1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 l L^–1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

Influence of Glyphosate on Nitrate Reductase Activity in Soybean (Glycine max)

Experiments were conducted to determine the influence of glyphosate[N-(phosphonomethyl)glycine] on extractable nitrate reductaseactivity during light and dark growth of soybean (Glycine max)seedlings. Glyphosate (5?10^–4 M), applied via root-feedingto three-day-old etiolated seedling, significantly reduced enzymeactivity in roots (48 to 96 h) and leaves (96 h) of seedlingsplaced in the light, but had little effect on enzyme activityin cotyledons compared to enzyme levels in tissues of untreatedseedlings. During dark-growth, nitrate reductase activity increasedwith time in cotyledons of untreated seedlings (activity about85-fold less than in cotyledons of light-grown plants) but muchlower enzyme levels were found in cotyledons of glyphosate-treatedseedlings after 72 and 96 h. In leaves of dark-grown seedlings,glyphosate reduced nitrate reductase levels by 95%. Most inhibitionof extractable enzyme activity occurred in newly developingorgans (leaves and roots) which correlates well with reportsthat glyphosate is rapidly translocated to these sites. However,the fact that glyphosate inhibits growth prior to lowering enzymeactivity levels indicates a secondary effect on nitrate reductase. (Received May 18, 1984; Accepted February 12, 1985)     

The Mode of Flower-Inhibiting Action of Ethylene in Pharbitis nil

The inhibitory effect of ethylene on photoperiodic flower inductionin Pharbitis nil was investigated in relation to the time ofethylene application. Ethylene applied during an 18-h dark periodnot only made the dark period non-inductive, but also greatlyinhibited the flower-inducing effect of the 2nd 18-h dark periodgiven 6 h after the end of the first dark period. The seconddark period was inductive when it was given 30 h after the endof the first dark period, during which ethylene was applied.Ethylene applied during the light period prior to an inductivedark period had no inhibitory effect, suggesting that ethylenegiven during the dark period produces some flower-inhibitingentity. (Received April 17, 1987; Accepted June 17, 1987)     

ATP Synthesis in Cotyledons of Cucumber and Mung Bean Seeds during the First Hours of Imbibition

ATP concentration increased rapidly during the first 6h of imbibitionin cotyledons of cucumber and mung bean seeds. The increasewas strongly inhibited by 1-h treatment of tissues with cyanidein both species. Carbonylcyanide m-chlorophenylhydrazone, anuncoupler of oxidative phosphorylation, showed little effectduring the first hour of the treatment, but its inhibitory effecton ATP synthesis became significant after 3 h. Mitochondrialfractions prepared from 6-h-old cucumber cotyledons were capableof phosphorylating ADP to ATP. These results suggest that mitochondrialoxidative phosphorylation may be involved in ATP synthesis duringthe early hours of imbibition in both cucumber and mung beanseeds. (Received December 7, 1987; Accepted April 9, 1988)     

The role of cotyledons in the establishment of Suaeda physophora seedlings

The role of cotyledons in seedling establishment of the euhalophyte Suaeda physophora under non-saline and saline conditions (addition of 1 mM or 400 mM NaCl) was investigated. Survival and fresh and dry weights were greater for seedlings grown in the light (12-h light/12-h dark) than in the dark (24-h dark). The shading of cotyledons tended to decrease shoot height, shoot organic dry weight, number of leaves, and survival of seedlings regardless of NaCl treatment, but the effect of cotyledon shading was greater with 400 mM NaCl. Concentrations of Na⁺ were higher in cotyledons than in leaves, regardless of NaCl treatment. The K⁺/Na⁺ ratio was lower in cotyledons than in leaves for seedlings treated with 1 mM NaCl but not for seedlings treated with 400 mM NaCl. Addition of 400 mM NaCl decreased oxygen production in cotyledons but especially in leaves. These results are consistent with the hypothesis that, by generating oxygen via photosynthesis and by compartmentalizing Na⁺, cotyledons are crucial for the establishment of S. physophora seedlings in saline environments.     

Changes in cotyledon mRNA during floral induction of Pharbitis nil CV. Violet

Floral induction in seedlings of Pharbitis nil Choisy cv. Violet, with one cotyledon removed, was manipulated by applying various photoperiodic treatments to the remaining cotyledon. Populations of polyadenylated RNA from treated cotyledons were examined to identify messages specifically involved in floral induction. The RNA was translated in vitro using a wheat-germ system, and the resulting translation products were analysed by two-dimensional polyacrylamide gel electrophoresis. Substantial qualitative and quantitative differences were found between mRNA from cotyledons of seedlings kept in continuous light (non-induced) and of seedlings given a 16-h dark period (induced). In contrast, inhibition of flowering with a night-break resulted only in one detectable, quantitative difference in mRNA.Abbreviations CL continuous light - kDa kilodalton - NB 16 h darkness+10 min red-light break, 8 h into the dark period - poly(A)⁺ RNA polyadenylated RNA (isolated by binding to a cellulose oligodeoxythymidine affinity column) - SD short day (16 h dark) - SDP short-day plant - SDS sodium dodecyl sulfate     

Flower-Inducing Activity of Phloem Exudates from Pharbitis cotyledons Exposed to Various Photoperiods

The phloem exudate prepared from the cotyledons of Pharbitisseedlings that had been exposed to a single dark period (oflonger than 10 h) induced flowering in cultured apices excisedfrom non-induced seedlings. The flower-inducing activity ofthe exudate increased as the seedlings were exposed to longerperiods of darkness. The highest activity was associated withthe exudate taken from cotyledons exposed to a single 16-h darkperiod. The activity of the exudate taken from cotyledons exposedto an inductive dark period was clearly reduced by interruptionof the dark period. The addition of exudate taken from threecotyledons to 10 ml of medium resulted in the highest flower-inducingactivity. About 50% of cultured apex explants formed floralbuds, even when the concentration of the exudate was reducedto 0.1 cotyledon equivalents per 10 ml of medium. The flower-inducingactivity of the exudate appeared to be heat-stable. (Received December 13, 1991; )     

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.     

Respiration Rate of Barley Roots: its Relation to Growth, Substrate Supply and the Illumination of the Shoot

The respiration rate of roots on intact barley plants grownin 16 h light 8 h dark cycles shows an exponential decay inthe dark, rises on re-illumination and there is a transientfall 12–14 h into the photoperiod Roots of plants placedin the dark for up to 48 h show a continued exponential decay,and a rather small fall in soluble carbohydrate levels The respirationof roots excised from predarkened plants does not rise on additionof sucrose to the medium bathing them Respiration rate, measured10 h into the photoperiod, shows a constant relation to rootweight in plants 8–24 days old, during which time rootcarbohydrate content first falls and later rises It is concludedthat root respiration rate is not a simple function of carbohydratesupply from the shoot The importance of root respiration inthe carbon budget of barley plants is evaluated and the levelsof control operating on root respiration rate are briefly discussed Hordeum distichum (L ) Lam, barley, respiration rate, light, carbohydrate     

Elimination of the Far-Red-Induced Lag Phase of Chlorophyll a Accumulation by a Brief Light in Pharbitis nil Cotyledons

In dark-grown Pharbitis nil seedlings, far-red light (FR) irradiationof 48 h or less promotes Chl a accumulation in the first 2-hof a subsequent white light (WL) period, without a lag phaseof Chl a accumulation. However, continuous FR irradiation of72 h or more, causes the so-called FR-induced lag phase. A 5-minWL given 4 h before the onset of the continuous WL promotesChl a accumulation irrespective of the length of the precedingFR irradiation period, if a 4-h dark period is inserted betweenthe 5-min WL and continuous WL. This suggests that the effectof the brief WL is independent of and additive to the effectof the preceding FR irradiation, although the effect of theFR irradiation changes from promotive to inhibitory with anincrease in the irradiation period. Red light (R) is more active than blue light (B) in this brieflight effect. The R effect is reversed by subsequent exposureto FR when the period of the preceding FR irradiation is 24h, but not when it is 72 h. The relative effectiveness of Bto R increases after prolonged FR irradiation. (Received August 6, 1986; Accepted March 12, 1987)     

Diurnal Fluctuations in Growth and CO2 Exchange in Dactylis glomerata

Diurnal fluctuations in dry matter accumulation and leaf extensionof seedlings of Dactylis glomerata were followed through a 16-hlight period and a subsequent 8-h dark period at 20 °C.Themeasured increase in dry weight during the light period andthe decrease during the dark period showed a very good agreementwith calculated dry weight changes derived from the rates ofcarbon dioxide exchange of whole seedlings. Although dry weightof the leaf blades decreased during the dark period, leaf expansioncontinued throughout the 24-h period with associated changesin the ratio of fresh weight to dry weight of the leaf blades.