Senescence and stomatal aperture as affected by antibiotics in darkness and light

In leaves of barley (Hordeum vulgare), as previously found with oats (Avena sativa), a group of six antibiotics that interfere in different ways with the sequence DNA → mRNA → protein all delay senescence in the dark, acting to conserve chlorophyll (Chl) and protein and also to open the stomata. Among the active compounds is chloramphenicol, which had previously been reported to act only on procaryotes. It is now shown that all these compounds with senescence-delaying action in darkness have the opposite effect in light, accelerating Chl destruction and partially or completely closing the stomata. Leaves of the dicot Tropaeolum majus show most of the same responses, though the changes in protein and amino acids are more variable. The data as a whole support the previous conclusion that the synthesis of one or more proteins controls both the opening and the closing of the stomata. An additional compound, kanamycin, acts in the same way as the other six compounds on oats and barley, though its action on proteolysis is unclear. On Tropaeolum, however, it opens the stomata in both light and darkness. Anisomycin and ethidium bromide have comparably atypical effects. Thus, although changes in stomatal opening or closing in the majority of cases are closely linked to the breakdown or preservation of Chl, the occasional exception shows that the biochemical phenomena of senescence cannot be under the direct control of changes in stomatal aperture.     

The interaction of hormonal and environmental factors in leaf senescence

The work concerns the senescence of isolated young leaves of oats (Avena sativa) floated on water or solutions. Senescence is rapid in darkness but slow in white light; the effect of light is not due to photosynthesis, but is paralleled by stomatal opening. Closure of the stomata by osmotic or chemical means makes senescence in light proceed as fast as in darkness, while opening the stomata in darkness by cytokinins, fusicoccin,etc., delays senescence to rates typical of light. The osmotic closure in light is mediated by abscisic acid, and since this also accumulates in darkness it appears as a major factor controlling senescence. Efflux of ions into the solution; indicating increased permeability, occurs almost in parallel with senescence. Senescence in light is accelerated by 1-aminocyclopropane-l-carboxylic acid (ACC) and inhibited by cobalt, silver or aminoethoxyvinyl glycine (AVG) which interfere with ethylene production or action; however, ethylene’s role is unclear because some reagents, including kinetin, that delay senescence, actually increase ethylene production. At the endogenous level, therefore, ethylene may not be a limiting factor. Finally, a new ethylene-generating system is described in which the dehydrogenation of linoleic acid is coupled through manganese to the oxidation of ACC; it is probably activein vivo.     

The Metabolism of Oat Leaves during Senescence: IV. The Effects of alphaalpha'-Dipyridyl and other Metal Chelators on Senescence

The senescence of the first leaves of light-grown Avena seedlings when detached and placed in the dark is inhibited by α, α′-dipyridyl and α, α′, α″-tripyridyl at concentrations between 10⁻⁵ and 10⁻⁴ M. Five other chelating agents exert similar inhibiting effects at concentrations 3 to 30 times higher. The senescence of etiolated leaves, as shown by loss of carotenoid and protein, is similarly inhibited. Ethylene-diaminetetraacetate has a similar effect in the dark, though only at 10 mM and above, but in the light it causes bleaching of chlorophyll. It is deduced that an iron-containing system plays an essential part in the initiation of the senescence process.     

Effects of light and spermine on senescence of hydrilla and spinach leaves

Light treatment markedly accelerated chlorophyll loss in Hydrilla (Hydrilla verticillata [L.f.] Royle) over dark treatment whereas such acceleration could not be observed in spinach (Spinacia oleracea L.) leaf segments. Spermine, a polyamine, retarded the loss of chlorophyll in the dark but markedly accelerated this loss in the light during senescence of Hydrilla leaves. However, such effect of spermine in the dark was not so pronounced in spinach. The loss of protein was slower in the light than in the dark in both the species. Spermine arrested the loss of protein (as in spinach) or even raised the protein level over initial (as in Hydrilla). Loss of both soluble and insoluble protein was slower in light than in darkness. Spermine treatment, either in light or darkness, markedly accelerated the loss of soluble protein but raised the level of insoluble protein over initial in both the species. The pattern of change in α-amino nitrogen in either species could be correlated well with that of protein level. In Hydrilla, light increased the soluble protein fraction over initial and this rise was prevented by cycloheximide and not by chloramphenicol. Also, spermine augmented the protease activity (both acid and neutral) while light retarded the rise in protease activity during senescence of either species. Although spermine treatment reduced the leaching of α-amino nitrogen and electrolytes in Hydrilla, it augmented the same in spinach.     

Light-induced h secretion and the relation to senescence of oat leaves

When abraded oat (Avena sativa L. cv Victory) leaf segments are floated on KCl solution, white light causes acidification of the solution external to leaf tissue. The presence of mannitol amplifies the light-induced proton secretion. Mature leaves as well as young ones acidify the medium in light, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in H⁺ secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H⁺ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent inhibitor of plasma membrane H⁺ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H⁺ secretion and acceleration of senescence, may suggest a regulatory role for H⁺ secretion in leaf senescence.     

Water Stress Reduces Ozone Injury via a Stomatal Mechanism

Various studies have shown that water-stressed plants are more tolerant of ozone exposures than are unstressed plants. Two probable explanations for this tolerance are (a) stomatal closure which reduces ozone uptake and (b) biochemical or anatomical changes within the leaves. Phaseolus vulgaris cv Pinto bean plants were established and transferred to membrane systems which controlled the osmotic potential around the roots at −35 or −80 kilopascals for 5 days prior to ozone treatment (0 or 1.0 microliters per liter for 2 hours). Both water-stressed and unstressed plants were sprayed with various concentrations of abscisic acid to close the stomata or with fusicoccin to induce stomata opening. The abaxial stomatal resistances of primary and trifoliate leaves were measured just prior to ozone exposure. Plant response to ozone was determined by stress ethylene production and chlorophyll loss. Both water stress and abscisic acid induced stomatal closure and reduced ozone injury. In water-stressed plants, fusicoccin induced stomatal opening and those plants were as sensitive to ozone as were the non-water-stressed plants. These data suggest that water stress protects plants from ozone injury mainly through its influence on stomatal aperture rather than through biochemical or anatomical changes.     

Amylases in Pea Tissues with Reduced Chloroplast Density and/or Function

Pea (Pisum sativum L.) tissues with reduced chloroplast density (e.g. petals and stems) or function (i.e. senescent leaves and leaves darkened for prolonged periods) were surveyed to determine whether tissues with genetically or environmentally reduced chloroplast density and/or function also have significantly different amylolytic enzyme activities and/or isoform patterns than leaf tissues with totally competent chloroplasts. Native PAGE followed by electrophoretically blotting through a starch or β-limit dextrin containing gel and KI/I₂ staining revealed that the primary amylases in leaves, stems, petals, and roots were the primarily vacuolar β-amylase (EC 3.2.1.2) and the primarily apoplastic α-amylase (EC 3.2.1.1). Among tissues of light grown pea plants, petals contained the highest levels of total amylolytic (primarily β-amylase) activity and considerably higher ratios of β- to α-amylase. In aerial tissues there was an inverse relationship between chlorophyll and starch concentration, and β-amylase activity. In sections of petals and stems there was a pronounced inverse relationship between chlorophyll concentration and the activity of α-amylase. Senescing leaves of pea, as determined by age, and protein and chlorophyll content, contained 3.8-fold (fresh weight basis) and 32-fold (protein basis) higher α-amylase activity than fully mature leaves. Leaves maintained in darkness for 12 days displayed a 14-fold (fresh weight basis) increase in α-amylase activity over those grown under continuous light. In senescence and prolonged darkness studies, the α-amylase that was greatly increased in activity was the primarily apoplastic α-amylase. These studies indicate that there is a pronounced inverse relationship between chloroplast function and levels of apoplastic α-amylase activity and in some cases an inverse relationship between chloroplast density and/or function and vacuolar β-amylase activity.     

Turnover of Fatty Acids during Natural Senescence of Arabidopsis, Brachypodium, and Switchgrass and in Arabidopsis β-Oxidation Mutants

During leaf senescence, macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds, or other storage organs. In this study, we determined the fatty acid levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon, and switchgrass (Panicum virgatum) leaves during natural senescence. In young leaves, fatty acids represent 4% to 5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced, resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 μg per leaf (0.6 μg mg⁻¹ dry weight). Triacylglycerol levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7, and kat2, which are blocked in enzyme activities of β-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and the wild type, indicating that several mutations in β-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.     

Is the effect of wounding on leaf senescence due to ethylene?

Wounding delays the loss of chlorophyll (Chl) that normally occurs when oat (Avena sativa L.) leaf segments are held in the dark. There was a continued increase in ethylene production during the senescence of the control segments; in contrast, ethylene production by the wounded segments, although it increased by a factor of 2–3 times, reached its peak in 48 h and then dropped sharply to below the basal level. Added 1-aminocyclopropane-1-carboxylic acid (ACC) caused a very large increase in ethylene production in both control and wounded segments, but it increased the rate of Chl loss, though only marginally. Aminoethoxyvinylglycine (AVG) inhibited ethylene production by both control and wounded segments and this did decrease the Chl loss, but only in the control segments. In the wounded segments, AVG antagonized the Chl-retaining action of the wound. Since wounding delayed the loss of Chl and yet caused a moderate increase in ethylene production, we conclude that the ethylene production by senescing oat leaves is not the main controlling influence in the wounding effect. The data also throw doubt on the causal participation of ethylene in normal Chl loss by these leaves in darkness.     

CYCLING OF STOMATAL APERTURE IN LEAVES OF PLANTS WITH CRASSULACEAN ACID METABOLISM UNDER CONSTANT CONDITIONS

When leaves of plants with C₃ metabolism are detached and held in darkness, they senesce and the stomata close. Because the relation of senescence and stomatal closure is very close, if not actually causal, the question arose as to whether in the leaves of plants with Crassulacean acid metabolism whose stomata open at night the relationship to senescence would be reversed. Detached leaves of four species of Hoya, floated on water in constant darkness or constant light, were found to show no large differences in stomatal aperture (measured as diffusion resistance) between those in the light or dark, but the aperture changed in a regular circadian rhythm. In some leaves the rhythm was simple, in others the peak showed small secondary peaks, but in all cases the values were nearly the same in the light as in the dark, throughout the cycle. Previous culture of the intact plants under normal day/night conditions gave results similar to those with plants that had had prolonged culture under constant light or darkness. In those cases when the stomata were more open in the dark, the chlorophyll content was greater than when the stomata were more open in the light; but when they were more open in the light, the chlorophyll content showed little difference between light and dark. When the leaves had only their petioles in water they showed greater senescence in the light than in the dark, and the stomata were more tightly closed in the light, especially at the apical ends. All four species of Hoya gave similar results. We deduce that senescence of these leaves is modified by stomatal aperture, and generally in the same direction as in C₃ leaves, but that in continuous light or darkness the primary control over the aperture is the endogenous cycle.     

Photosynthesis at High Temperature in Tuber-Bearing Solanum Species : A Comparison between Accessions of Contrasting Heat Tolerance

Differences in the photosynthetic performance between pairs of heat tolerant (HT) and heat sensitive (HS) accessions of tuber-bearing Solanum species were measured at 40 °C, after treating plants at 40/30 °C. After 1 to 9 days of heat treatment, both HT and HS accessions showed progressive inhibitory effects, primarily decreased rates of CO₂ fixation, and loss of leaf chlorophyll. These effects were most pronounced in the HS accessions. Stomatal conductivity and internal CO₂ concentrations were lower for both accessions at 40 °C especially for the HS accessions, suggesting that at ambient CO₂ concentrations, stomatal conductance was limiting CO₂ availability at the higher temperature. In the HT accessions, stomatal limitations were largely attributed to differences in vapor pressure deficit between 25° and 40 °C, while the HS accessions exhibited significant nonstomatal limitations. The young expanding leaves of the HS accession showed some HT characteristics, while the oldest leaves showed severe senescence symptoms after 9 days at 40/30 °C. The data suggest that differences in heat sensitivity between HT and HS accessions are the result of accelerated senescence, chlorophyll loss, reduced stomatal conductance, and inhibition of dark reactions at high temperature.     

Correlation of leaf senescence and gene expression/activities of chlorophyll degradation enzymes in harvested Chinese flowering cabbage (Brassica rapa var. parachinensis)

Chinese flowering cabbage is one of the main leafy vegetables produced in China. They have a rapid leaf yellowing due to chlorophyll degradation after harvest that limits their marketing. In the present study, leaf senescence of the cabbages was manipulated by ethylene and 6-benzyl aminopurine (6-BA) treatment to investigate the correlation of leaf senescence and chlorophyll degradation related to gene expression/activities in the darkness. The patterns of several senescence associated markers, including a typical marker, the expression of senescence-associated gene SAG₁₂, demonstrated that ethylene accelerated leaf senescence of the cabbages, while 6-BA retarded this progress. Similar to the trends of BrSAG₁₂ gene expression, strong activation in the expression of three chlorophyll degradation related genes, pheophytinase (BrPPH), pheophorbide a oxygenase (BrPAO) and red chlorophyll catabolite reductase (BrRCCR), was detected in ethylene treated and control leaves during the incubation, while no evident increase was recorded in 6-BA treated leaves. The overall dynamics of Mg-dechelatase activities in all treatments displayed increasing trends during the senescence process, and a delayed increase in the activities was observed for 6-BA treated leaves. However, chlorophyllase activity as well as the expression of BrChlase1 and BrChlase2 decreased with the incubation in all treatments. Taken together, the expression of BrPPH, BrPAO and BrRCCR, and the activity of Mg-dechelatase was closely associated with the chlorophyll degradation during the leaf senescence process in harvested Chinese flowering cabbages under dark conditions.     

Development of chlorophyll and hill activity

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding δ-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with δ-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.     

Effects of sulfur on the photosynthesis of intact leaves and isolated chloroplasts of sugar beets

Effects of sulfur on photosynthesis in sugar beets (Beta vulgaris L. cv. F58-554H1) were studied by inducing sulfur deficiency and determining changes in the photosynthesis of whole attached leaves and of isolated chloroplasts. The rates of photosynthetic CO₂ uptake by intact leaves, photoreduction of ferricyanide, cyclic and noncyclic photophosphorylation of isolated chloroplasts, and the rate of CO₂ assimilation by ribulose diphosphate carboxylase, decreased with decrease in total leaf sulfur from 2500 to about 500 μg g⁻¹ dry weight. Sulfur deficiency reduced photosynthesis through an effect on chlorophyll content, which decreased linearly with leaf sulfur, and by decreasing the rate of photosynthesis per unit chlorophyll. There was only a small effect of sulfur deficiency on stomatal diffusion resistance to CO₂ until leaf sulfur decreased below 1000 μg g⁻¹ when stomatal resistance became a more significant proportion of the total diffusion resistance to CO₂. Light respiration rates were positively correlated with photosynthesis rates and dark respiration was unchanged as leaf sulfur concentrations declined.     

Stomata Prioritize Their Responses to Multiple Biotic and Abiotic Signal Inputs

Stomata are microscopic pores in leaf epidermis that regulate gas exchange between plants and the environment. Being natural openings on the leaf surface, stomata also serve as ports for the invasion of foliar pathogenic bacteria. Each stomatal pore is enclosed by a pair of guard cells that are able to sense a wide spectrum of biotic and abiotic stresses and respond by precisely adjusting the pore width. However, it is not clear whether stomatal responses to simultaneously imposed biotic and abiotic signals are mutually dependent on each other. Here we show that a genetically engineered Escherichia coli strain DH5α could trigger stomatal closure in Vicia faba, an innate immune response that might depend on NADPH oxidase-mediated ROS burst. DH5α-induced stomatal closure could be abolished or disguised under certain environmental conditions like low [CO₂], darkness, and drought, etc. Foliar spraying of high concentrations of ABA could reduce stomatal aperture in high humidity-treated faba bean plants. Consistently, the aggressive multiplication of DH5α bacteria in Vicia faba leaves under high humidity could be alleviated by exogenous application of ABA. Our data suggest that a successful colonization of bacteria on the leaf surface is correlated with stomatal aperture regulation by a specific set of environmental factors.     

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O₂ and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and α-ketoglutarate) in CO₂-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO₂-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO₂-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.     

Metabolism of Oat Leaves during Senescence : VIII. The Role of L-Serine in Modifying Senescence

The mechanism whereby l-serine specifically promotes the dark senescence of detached oat (Avena) leaves has been examined. The fact that this promotion is strong in darkness but very weak in white light has been explained, at least in part, by the finding that added serine is partly converted to reducing sugars in light. Labeled serine gives rise to ¹⁴C-sugars and ¹⁴CO₂. In the absence of CO₂, serine does cause chlorophyll loss in light and undergoes a decreased conversion to sugar.     

Apple leaf senescence: leaf disc compared to attached leaf

Attached apple leaves (Pyrus malus L., Golden Delicious) began to lose protein in early August as the first sign of senescence. Apple leaf discs prepared from samples before early August gained protein for up to 7 days after detachment. After early August, the loss of protein from leaf discs was no greater than the loss from attached leaves in 7 days. The loss of chlorophyll from leaf discs began over 2 months before attached leaves began to lose chlorophyll naturally and before leaf discs lost protein. Leaf discs from presenescent leaves did not senesce significantly faster when maintained in darkness instead of 12 hours of light. In general, the loss of protein and chlorophyll from apple leaf discs after 7 days was much less than for most other leaf types studied.     

Senescence-Associated Changes during Long-day-induced Leaf Senescence and the Nature of the Graft-transmissible Senescence Substance in Kleinia articulata

Schwabe, W. W. and Kulkarni, V. J. 1987. Senescence-associatedchanges during long-day-induced leaf senescence and the natureof the graft-transmissible senescence substance in Kleinia articulata.— J. exp. Bot. 38: 1741–1755. The long-day-induced senescence in Kleinia articulata leaveswas characterized by a loss in fresh and dry weight, in therate of leaf expansion and progressive loss of chlorophyll inthe detached rooted leaves. Ultrastructural examination of mesophyllcells of leaves from plants grown in continuous light showedthat osmiophilic globules accumulating in the chloroplasts werethe first visible sign of senescence in the organdies. Thesefirst signs of senescence could be detected in very young leavesof plants in continuous light, even before the leaves had expanded.Attempts were made to study the cause of this photoperiodicsenescence which, from previous work, appeared to involve agraft-transmissible substance. Leaves in continuous light showed reduced stomatal opening andextracts from them had very much higher activity in the Commelinastomatal closure assay (ABA-like activity ?) compared with non-senescingleaves grown in short days (8 h). However, even if all the activitywere due to ABA, this on its own does not appear to be the senescencesubstance because a much longer exposure to continuous lightwas required to induce irreversible senescence than to reachmaximum stomatal closure promoting activity in the bioassay.Moreover, severe water stress (high ABA?) did not lead to senescenceunless combined with continuous light or ethylene treatment.It is postulated that while ABA may play an important role inKleinia leaf senescence its lethal effect may not be realizedunless ethylene-induced membrane changes may synergisticallyassist. Key words: Leaf senescence, ABA, Daylength, stomatal movement, Kleinia