Differential changes in the electron transport properties of thylakoid membranes during aging of attached and detached leaves, and of isolated chloroplasts

Abstract. Aging of chloroplasts both in vivo and in vitro causes a considerable loss in the 2,6-dichlorophenol indophenol (DCPIP)-Hill reaction with water as electron donor. The loss can be reduced by exogenous electron donors like diphenyl carbazide (DPC). suggestive of aging-induced damage of the oxygen evolving system. Aging also brings about a considerable loss in methylviologen (MV) reduction mediated by Photosystem I (PS I) of chloroplasts with an ascorbate-DCPIP couple as the electron donating system.
The loss in the electron transport ability of the plastids is faster during in vitro compared to in vivo aging of the chloroplasts.
Light protects the photo-electron transport ability of chloroplasts during aging of intact leaves in contrast to its action during aging of the isolated organelles.     

Influence of Aging on Nuclear DNA in Corn Leaves

Leaves of Zea mays L. were selected at a young stage when theyhad accumulated about two-thirds of their maximum chlorophyll,at a mature stage when approximately full dimensions and maximumchlorophyll levels were attained, and following pollen shedwhen lower leaves were dying from the tip. Data show a lossof one-fourth of the nuclear DNA, no effect of aging on eu-and heterochromatin levels and a marked decline in the guanine(G) and cytosine (C) percentages. Although DNA levels mightbe under hormonal control, it is not clear that the loss detectedwould be physiologically influential in aging sequences. Yetthis decline preceded those of leaf nitrogen and chlorophyll. (Received July 7, 1983; Accepted November 23, 1983)     

Response of senescing rice leaves to flooding stress

Flooding stress (FS) induced changes in pigment and protein contents and in photochemical efficiency of thylakoid membranes of chloroplasts were investigated during senescence of primary leaves of rice seedlings. Leaf senescence was accompanied by loss in 2,6-dichlorophenolindophenol (DCPIP) photoreduction, rate of oxygen evolution, quantum yield of photosystem 2 with an increase in MDA accumulation, and non-photochemical quenching (NPQ) of chlorophyll fluorescence. These changes were further aggravated when the leaves during this period experienced FS. The increase in NPQ value under stress may indicate photosynthetic adaptation to FS.     

Effect of phytohormones on chlorophyll degradation during aging of chloroplastsin vivo andin vitro

Summary Phytohormones like IAA and kinetin inhibit chlorophyll loss during aging of wheat chloroplasts duringin vivo andin vitro. GA, on the other hand, stimulates the pigment degradation during aging of attached leaves in contrast to its senescence inhibiting action in detached leaves and isolated chloroplasts. A shift in optimum concentration of hormone in inhibiting chlorophyll degradation suggests a differential pool size of endogenous hormone regulating aging of chloroplastsin vivo andin vitro. The retardation of chlorophyll loss by kinetin, IAA and GA during aging of chloroplastsin vitro would indicate that the effect of hormones in preventing yellowing of senescing leaves may be mediated through their direct action on chloroplasts.     

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.     

Changes occurring during aging and senescence in a submerged aquatic angiosperm (Potamogeton pectinatus)

Changes occurring during aging and senescence of leaves of a submerged aquatic angiosperm ( Potamogeton pectinatus L.) were studied. Total chlorophyll and chlorophylls a and b were maximal in mature, and minimal in old leaves. The chlorophyll a to b ratio was highest in mature leaves. During senescence, the chlorophyll content and the ratio of chlorophyll a to b decreased. The content of DNA, RNA, protein and dry weight, and the activity of alkaline pyrophosphatase decreased while free amino acids, the activity of protease, RNase and acid pyrophosphatase, and the ratio of acid to alkaline pyrophosphatase activity increased during aging and senescence. Kinetin (0.23 m M ) deferred leaf senescence by delaying the loss of chlorophyll, protein, nucleic acids and dry weight, and reducing the rise in free amino acids, the activity of protease, RNase and acid pyrophosphatase and the ratio of acid to alkaline pyrophosphatase activity; while both 0.69 m M ethrel and 0.075 m M ABA hastened senescence. Kinetin pretreatment for an optimum period (12 h) followed by ethrel or ABA treatment partially erased the senescence-promoting effect of the latter. But treatments in a reverse order markedly reduced the delaying effect of kinetin on senescence.     

Reversine ameliorates hallmarks of cellular senescence in human skeletal myoblasts via reactivation of autophagy

Cellular senescence leads to the depletion of myogenic progenitors and decreased regenerative capacity. We show that the small molecule 2,6-disubstituted purine, reversine, can improve some well-known hallmarks of cellular aging in senescent myoblast cells. Reversine reactivated autophagy and insulin signaling pathway via upregulation of Adenosine Monophosphate-activated protein kinase (AMPK) and Akt2, restoring insulin sensitivity and glucose uptake in senescent cells. Reversine also restored the loss of connectivity of glycolysis to the TCA cycle, thus restoring dysfunctional mitochondria and the impaired myogenic differentiation potential of senescent myoblasts. Altogether, our data suggest that cellular senescence can be reversed by treatment with a single small molecule without employing genetic reprogramming technologies.     

Control of photosynthetic sucrose synthesis in barley primary leaves: role of fructose 2,6-bisphosphate

Levels of fructose 2,6-bisphosphate (F2,6BP) and related metabolites were measured in 8- or 9-day-old barley (Hordeum vulgare L.) primary leaves throughout a 24 hour cycle. Young barley leaves contained about 0.4 nanomole F2,6BP per milligram chlorophyll at the end of a 12 hour dark period. F2,6BP levels increased rapidly following a dark-to-light transition and then decreased to about 0.1 nanomole per milligram chlorophyll after 5 or 10 minutes of light. Low levels of F2,6BP were detected in barley primary leaves throughout the day. A 10-fold increase in F2,6BP was observed during the first hour of the dark period and then levels of this metabolite decreased slowly for the next several hours. Only small diurnal fluctuations were noted in barley leaf glucose 6-phosphate and uridine 5′-diphosphoglucose levels. There were rapid changes in whole leaf F2,6BP levels when the light intensity was altered. High F2,6BP levels in the dark were not observed after short photosynthetic periods. Results obtained with barley primary leaves support the suggestion that F2,6BP is involved in regulating the flow of photosynthate from the chloroplast to sucrose. Extractable sucrose-phosphate synthase activity was inversely related to barley primary leaf F2,6BP levels. This finding may indicate that the activities of sucrose-phosphate synthase and cytosolic fructose 1,6-bisphosphatase in barley primary leaves are metabolically coordinated.     

Fructose 2,6-bisphosphate levels in mature leaves of tobacco (Nicotiana tabacum) and potato (Solanum tuberosum)

Here we show that fructose 2,6-bisphosphate cannot be reliably measured in mature leaves of tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.), or stinging nettle (Urtica dioica L.) using conventional extraction techniques, since the recoveries of fructose 2,6-bisphosphate added during extraction are poor. However, fructose 2,6-bisphosphate could be extracted by boiling leaves in ethanol and aqueous buffer. Evidence for the reliability of this technique is provided by high recovery measurements of fructose 2,6-bisphosphate added to the leaves before extraction. This extraction method was used to measure changes in the level of fructose 2,6-bisphosphate throughout the photoperiod in tobacco and potato leaves. These changes are compared with the rate of accumulation of sucrose and starch in the leaf samples. Variations in the levels of fructose 2,6-bisphosphate, and the relationship between this metabolite and sucrose and starch accumulation in these leaves during the photoperiod are similar to the pattern observed in leaves of other plant species.Abbreviations BSA bovine serum albumin - Fru-2,6-P₂ fructose 2,6-bisphosphate This research was supported by the Agricultural and Food Research Council (Grant no. PG43/531), and the Royal Society.     

Compartment Analysis of Nitrogen Flows through Mature Leaves

Nitrogen flows in mature leaves of rice, sunflower and cornwere analyzed by multicompartment analyses of data from ¹⁵Ntracer experiments. The fourcompartment model fit the measureddata better than the two- or three-compartment models. Rateconstants of the nitrogen flows, transfers with or without proteinturnover and flow through a storage pool of soluble-N, werederived from a least-squares fit between the mathematical expressionsand the corresponding measured data. Results of computations indicated the following: The flow ofnitrogen through the protein pool was larger than the directflow in recently matured leaves, whereas the reverse was truein senescent leaves. The presence of a temporary storage poolof soluble N was suggested, and the half-life of the N exchangein that pool was estimated to be roughly the amount of the insolubleN present in three of the four cases examined. The half-livesof insoluble N (protein) pools were 35 to 150 h. The pool forthe N efflux from the leaves was small with the shortest half-lives(most less than 2 h) of N turnover. Some recycling of N duringprotein turnover was suggested in sunflower leaves, but theextent in the leaves of rice seedlings was estimated to be little. (Received April 28, 1983; Accepted October 6, 1983)     

Contribution of different protein phosphatases to the dephosphorylation of 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in rat liver.

The nature of rat liver protein phosphatases involved in the dephosphorylation of the glycolytic key enzyme 6-phosphofructo-1-kinase and the regulatory enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase was investigated. In terms of the classification system proposed by Ingebritsen & Cohen [(1983) Eur. J. Biochem. 132, 255-261], only the type-2 protein phosphatases 2A (which can be separated into 2A1 and 2A2) and 2C act on these substrates. Fractionation of rat liver extracts by anion-exchange chromatography and gel filtration revealed that protein phosphatase 2A is responsible for most of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase phosphatase activity (activity ratio 2A/2C = 4:1). On the other hand, 6-phosphofructo-1-kinase phosphatase activity is equally distributed between protein phosphatases 2A (2A1 plus 2A2) and 2C. In addition, the possible role of low-Mr compounds for the control of purified protein phosphatase 2C was examined. At near-physiological concentrations, none of the metabolites studied significantly affected the rate of dephosphorylation of 6-phosphofructo-1-kinase, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, pyruvate kinase or fructose-1,6-bisphosphatase.     

Separation of Chlorophyll Degradation from Other Senescence Processes in Leaves of a Mutant Genotype of Meadow Fescue (Festuca pratensis L.)

Chlorophyll levels in l-cm sections of the youngest fully expanded leaves of normal (Y) Festuca pratensis L. declined almost to zero over a period of 6 days after excision. Chlorophyll in a mutant genotype (NY) remained near the initial level for the whole of this period. Abscisic acid promoted pigment loss in Y but had no significant effect on chlorophyll in NY. Kinetin retarded pigment loss in Y but was ineffective in NY. Other biochemical changes associated with leaf senescence—reduction in protein content and the appearance of novel isoenzymes of α-naphthyl acetate esterases—occurred in both genotypes. Abscisic acid accelerated protein breakdown, whereas kinetin inhibited the loss of protein in both genotypes. The mutation thus appears to be expressed as a highly specific lesion in pigment metabolism. We concluded that pigment breakdown, which is widely used as an index of leaf senescence, may not be an inevitable part of the aging process.     

Photosynthetic carbohydrate metabolism in wheat (Triticum aestivum L.) leaves: optimization of methods for determination of fructose 2, 6-bisphosphate

The accurate measurement of fructose 2,6-bisphosphate from plants such as wheat is fraught with difficulty. Extraction and assay methods for fructose 2,6-bisphosphate that give near 100% recovery of the metabolite, and a linear response with volume have therefore been developed for extracts prepared from wheat leaves of different ages. Amounts of fructose 2,6-bisphosphate in different regions of leaves generally showed a positive correlation with chlorophyll content. Measurements of sucrose and starch in third leaves harvested at different times of the diurnal cycle demonstrated that sucrose is the major form in which photosynthate is stored in the leaf, but starch can account for up to about 30% of the stored carbohydrate. Virtually all of the carbohydrate accumulated as starch and sucrose during the day was degraded at night. Amounts of fructose 2,6-bisphosphate were generally lower in extracts prepared from leaves harvested in the light than in the dark. Additionally, there was no change in either the amount of fructose 2, 6-bisphosphate or the ratio of sucrose to starch in samples prepared from leaves harvested at different times of the day. These results are broadly consistent with a role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis and the partitioning of carbohydrate between sucrose and starch in wheat leaves.     

Kinetic properties of bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from spinach leaves.

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. The soluble protein had a molecular mass of 320 kDa, estimated by gel filtration chromatography, and a subunit size of 90.8 kDa. The purified protein had activity of both 6-phosphofructo-2-kinase specific activity 10.4-15.9 nmol min(-1) x mg protein (-1) and fructose-2,6-bisphosphatase (specific activity 1.65-1.75 nmol x mol(-1) mg protein(-1). The 6-phosphofructo-2-kinase activity was activated by inorganic phosphate, and inhibited by 3-carbon phosphorylated metabolites and pyrophosphate. In the presence of phosphate, 3-phosphoglycerate was a mixed inhibitor with respect to both fructose 6-phosphate and ATP. Fructose-2,6-bisphosphatase activity was sensitive to product inhibition; inhibition by inorganic phosphate was uncompetitive, whereas inhibition by fructose 6-phosphate was mixed. These kinetic properties support the view that the level of fructose 2,6-bisphosphate in leaves is determined by the relative concentrations of hexose phosphates, three-carbon phosphate esters and inorganic phosphate in the cytosol through reciprocal modulation of 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities of the bifunctional enzyme.     

Influence of Elevated Fructose-2,6-Bisphosphate Levels on Starch Mobilization in Transgenic Tobacco Leaves in the Dark

The aim of this work was to study the effect of elevated fructose-2,6-bisphosphate (Fru-2,6-bisP) levels on carbohydrate metabolism in leaves in the dark. In transgenic tobacco (Nicotiana tabacum L.) lines containing mammalian 6-phosphofructo-2-kinase activity there is an inverse relationship between the level of Fru-2,6-bisP in leaves and the rate of starch breakdown in the dark. Estimates of the flux response coefficient for the rate of net starch degradation with respect to changes in Fru-2,6-bisP level are -0.57 for whole leaves and -0.69 to -0.89 for excised leaf discs. We suggest that this decrease in the net rate of starch breakdown is caused, at least in part, by stimulation of unidirectional starch synthesis. Measurements of the levels of metabolic intermediates and the metabolism of [U-14C]glucose indicate that the stimulation of starch synthesis in the dark is a result of high Fru-2,6-bisP levels, increasing the 3-phosphoglycerate:inorganic phosphate ratio in leaves. We argue that the observed response to changes in the level of Fru-2,6-bisP are effected through activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, the extent to which changes in Fru-2,6-bisP influence starch metabolism in wild-type plants is not known.     

A comparative analysis of fructose 2,6-bisphosphate levels and photosynthate partitioning in the leaves of some agronomically important crop species

Starch, sucrose, and fructose 2,6-bisphosphate (F2, 6BP) levels were measured in pea (Pisum sativum L.), maize (Zea mays L.), onion (Allium cepa L.) and soybean (Glycine max L.) leaves throughout a light/dark cycle. Leaf starch accumulated in pea, maize, and soybean but not in onion. Sucrose was a major leaf storage reserve in pea, maize, and onion but was only found at low levels in soybean. In all species examined, the most dramatic changes in F2,6BP concentration coincided with light/dark transitions. During the light period F2,6BP levels were about 0.1 nanomole/milligram chlorophyll in soybean source leaves and there was a small increase in effector concentration in the dark. Levels of F2,6BP were also low in pea and maize leaves during the light period but then increased 10- or 20-fold in the dark. Dark onion leaf F2,6BP levels were about 1.1 to 1.3 nanomole/milligram chlorophyll and these values decreased by 20 to 30% in the light. Thus, three different patterns were identified that describe diurnal F2,6BP levels in source leaves. These results support the suggestion that F2,6BP is involved in the regulation of sucrose biosynthesis. However, it was not possible to demonstrate that high levels of F2,6BP are essential for starch synthesis in the chloroplast.     

Water relations of cotton plants under nitrogen deficiency

Nitrogen deficiency in cotton plants (Gossypium hirsutum L.) increased the threshold water potentials for both stomatal closure and leaf senescence (defined as loss of chlorophyll and protein) during drought. These studies attempted to answer two questions: (1) What is the basis for the N/water interaction on senescence? (2) Is there a direct relationship between stomatal closure and senescence? Young and old leaves from N-deficient and N-sufficient plants maintained their relative sensitivities to water stress when excised leaf discs were floated on solutions of polyethylene glycol in dim light. In this leaf disc system, both leaf aging and N deficiency increased the threshold water potential for senescence. Leaf aging and N deficiency also decreased the concentration of exogenous abscisic acid necessary to initiate senescence in discs. A role for cytokinins in controlling senescence could not be clearly shown. In young leaves of both N-deficient and N-sufficient plants, stomata closed at water potentials much higher than those causing senescence. During leaf aging, the water potentials causing senescence increased more than those causing stomatal closure. The two processes thus occurred at about the same potentials in the oldest leaves. These data argue against a general cause-and-effect relationship between stomatal closure and senescence. Rather, each process apparently responded independently to absicsic acid accumulated during drought.     

Phosphorylation of a bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase, is regulated physiologically and developmentally in rosette leaves of Arabidopsis thaliana.

The phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (EC 2.7.1.105/ EC 3.1.3.46) in rosette leaves of Arabidopsis was examined. Immunoblotting with specific antisera detected 96-kDa and 92-kDa bands in the crude protein extracts from rosette leaves of Arabidopsis. Incubation of protein samples with alkaline phosphatase before SDS-PAGE reduced the 96-kDa band with concomitant increase of the 92-kDa band, suggesting that the former is a phosphorylated form of the latter. In accordance with this result, 96-kDa and 92-kDa bands were immuno-precipitated from the crude protein extracts from [(32)P]orthophosphate-labeled rosettes of Arabidopsis; and, the former was heavily labeled, the latter faintly labeled. Analysis of phospho-amino acid residues derived from the [(32)P]-labeled 96-kDa band revealed that the phosphorylation occurred on serine and threonine residues, excluding the possibility that the phosphorylated band represent a phospho-histidine intermediate that is known to form in the phosphatase reaction. The relative level of the 96-kDa band over the 92-kDa band in whole rosette extracts changed diurnally and was highest at the beginning of nighttime. Furthermore, the 96-kDa band was highly enriched in the extracts of very young rosette leaves, suggesting that the phosphorylation status of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase is regulated physiologically and developmentally in Arabidopsis.     

Quantitative Aspects of the in Vivo Regulation of Pyrophosphate:Fructose-6-Phosphate 1-Phosphotransferase by Fructose-2,6-Bisphosphate

Pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was quantified in developing barley (Hordeum vulgare) leaves by immunostaining on western blots using a purified preparation of barley leaf PFP as standard. Fructose-2,6-bisphosphate (Fru-2,6-bisP) was quantified in the same tissues. Depending on age and tissue development, the concentration of PFP varied between 11 and 80 [mu]g PFP protein g-1 fresh weight, which corresponds to 0.09 to 0.65 nmol g-1 fresh weight of each of the [alpha] and [beta] PFP subunits. The level depends primarily on the maturity of the tissue. In the same tissues the concentration of Fru-2,6-bisP varied between 0.07 and 0.46 nmol g-1 fresh weight. Thus, the concentrations of PFP subunits and Fru-2,6-bisP were of the same order of magnitude. In young leaf tissues the concentration of PFP subunits may exceed the concentration of Fru-2,6-bisP. This means that the amount of Fru-2,6-bisP present will be too low to occupy all the allosteric binding sites on PFP even though the concentration of Fru-2,6-bisP exceeds the Ka(Fru-2,6-bisP) by several orders of magnitude. These results are discussed in relation to Fru-2,6-bisP as a regulator of enzyme activities under in vivo conditions.     

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.