Senescence mechanisms

Senescence in plants is usually viewed as an internally programmed degeneration leading to death. It is a developmental process that occurs in many different tissues and serves different purposes. Generally, apoptosis refers to programmed death of small numbers of animal cells, and it shows some special features at the cell level. Some senescing plant cells show some symptoms typical of apoptosis, while others do not. This review will focus primarily on leaf senescence with ultimate aim of explaining whole plant senescence (i.e., monocarpic senescence). Traditionally, the ideas on senescence mechanisms fall into two major groupings, nutrient deficiencies (e.g., starvation) and genetic programming (i.e., senescence-promoting and senescence-inhibiting genes). Considerable evidence indicates that nutrient deficiencies are not central senescence program components, while increasing evidence supports genetic programming. Because chlorophyll (Chl) and chloroplast (CP) breakdown are so prominent, leaf senescence is generally measured in terms of Chl loss. Although CP breakdown may not be the proximate cause of leaf cell death, it certainly is important as a source of nutrients for use elsewhere, e.g., for developing reproductive structures in monocarpic plants, and this loss limits assimilatory capacity. The CP is dismantled in an orderly sequence. Individual protein complexes seem to be taken out all at once, not one subunit at a time. Removal of any component, e.g., Chl, seems to destabilize the whole complex. It is of special interest that senescing CPs secrete Chl-containing globules indicating that some CP components are broken down outside the CP. Senescence appears to be imposed on the CP by the nucleus, and all the known senescence-altering genes except one, cytG in soybean, are nuclear. Only the d₁d₂ mutation(s) in soybean prevents a broad range of leaf senescence processes. Exactly, what causes cell death is unclear; however, the selective thiol protease inhibitor, E-64, does delay death, and this suggests that proteases play a key role.     

Peroxidase and Senescence

Kinetin and a, á-dipyridyl prevented the rapid decreaseof chlorophyll content in detached oat leaves senescing in thedark. In the light, detachment caused a 27–40% rise in peroxidaseactivity and kinetin enhanced the enzyme in the segments byabout 80%. Darkness prevented any detachment-induced rise ofthe activity and decreased the stimulating action of kinetinand mechanical injury. The effect of dipyridyl on peroxidaseactivity in the dark was similar to that of kinetin. Kinetin enhanced the same distinctive isoperoxidases under lightand dark conditions. Neither horseradish peroxidase nor that extracted from oat leavesshowed any ability to hydroxylate free proline in vitro. A systemwhich supposedly led to peroxidase-catalysed proline hydroxylationyielded small amounts of hydroxyproline in the absence of theenzyme. Staining with Fast Blue BB salt in the presence of IAA as asubstrate after electrophoresis indicated that all detectedoat isoperoxidases had an IAA oxidase activity visually parallelingtheir peroxidase activity. Crude extracts contained IAA oxidaseinhibitors that could be partially or fully removed by dialysis. The possible significance of the rise in peroxidase activityduring senescence is discussed.     

Senescence of Rice Leaves XVIII. Changes of Stomatal Aperture during Senescence

Changes of stomatal aperture during the course of developmentof rice leaves were directly observed with a scanning electronmicroscope. The stomata reached their maximal aperture sizeafter senescence began in seedling leaves and the flag leafof mature plants. The small stomatal aperture observed priorto senescence seems to be the normal size of stomata in riceleaves, and thus stomata closure does not seem to be the causeof leaf senescence in rice plants. The stomata retain theircapability of movement during senescence, suggesting that guardcells tend to live longer than mesophyll cells. ⁴Present address: Tobacco Taiwan, Republic of China ResearchInstitute, Taichung, Taiwan, Republic of China (Received March 12, 1987; Accepted September 30, 1987)     

Metabolism of Oat Leaves during Senescence: V. Senescence in Light

A comparison has been made of the progress of senescence in the first leaf of 7-day-old oat plants (Avena sativa cv. Victory) in darkness and in white light. Light delays the senescence, and intensities not over 100 to 200 ft-c (1000-2000 lux) suffice for the maximum effect. In such intensities, chlorophyll loss and amino acid liberation still go on in detached leaves at one-third to one-half the rate observed in darkness; however, when the leaves are attached to the plant, the loss of chlorophyll in 5 days is barely detectable. Transfer of the leaves from 1 or 2 days in the low intensity light to darkness, or vice versa, shows no carryover of the effects of the preceding exposure, so that such treatment affords no evidence for the photoproduction of a stable substance, such as cytokinin, inhibiting senescence. Light causes a large increase in invertaselabile sugar and a smaller increase in glucose, and application of 100 to 300 mm glucose or sucrose in the dark maintains the chlorophyll, at least partially. Correspondingly, short exposure to high light intensity, which increased the sugar content, had a moderate effect in maintaining the chlorophyll. However, 3-(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU) completely prevents the increases in sugars and yet does not prevent the effect of light on senescence, whether determined by chlorophyll loss or by protein hydrolysis. Light causes a 300% increase in the respiration of detached oat leaves, and kinetin lowers that only partly, but unlike the increased respiration associated with senescence in the dark, the increase in the light is fully sensitive to dinitrophenol, and therefore cannot be ascribed to respiratory uncoupling. The increased respiration in light is prevented by DCMU, parallel with the prevention of sugar formation. It is therefore ascribed to the accumulation of soluble sugars, acting as respirable substrate. Also, l-serine does not antagonize the light effect. For all of these reasons, it is concluded that the action of light is not mediated by photosynthetic sugar formation, nor by photoproduction of a cytokinin. Instead, we propose that light exerts its effect by photoproduction of ATP. The action of sugars is ascribed to the same mechanism but by way of respiratory ATP. This hypothesis unifies most of the observed phenomena of the senescence process in oat leaves, and helps to explain some of the divergent findings of earlier workers.     

Senescence of Detached Fern Leaves

Detached fern leaves show a loss in the content of pigments,protein and a decline in 2,6-dichlorophenol indophenol Hillreaction of isolated chloroplasts during aging. Light treatmentof leaves could considerably retard aging induced loss in 2,6-dichlorophenolindophenol photoreduction but it exhibits less effect on pigmentand protein loss. (Received February 7, 1983; Accepted July 14, 1983)     

The Metabolism of Oat Leaves during Senescence: III. The Senescence of Isolated Chloroplasts

The changes in chlorophyll and protein in senescing chloroplasts isolated from the first leaves of 7-day-old oat (Avena sativa) seedlings have been investigated. In darkness the chlorophyll in these plastids is highly stable, losing only 5 to 10% of its content after 7 days at 26 C. This result contrasts with the behavior of chlorophyll in intact leaves, in which about 80% of the pigment would have disappeared in that time. The protein is less stable than the chlorophyll, though more stable than in the leaf; probably a small amount of protease is present in the plastids. Some protein is also being synthesized in the chloroplasts along with its breakdown; gains of up to 38% in protein and 13% in chlorophyll were observed under different conditions. l-Serine, which actively promotes senescence in the leaf, has only a very slight effect on the chloroplasts, and kinetin antagonizes it. Kinetin also has a small but significant effect in preserving the protein from breakdown. Acid pH somewhat promotes the breakdown, both of chlorophyll and protein. A loss of chlorophyll and protein comparable to that occurring in the senescence of the leaf could not be induced in the chloroplasts by suspending them in malate, in cytoplasmic extract, or in any of a number of enzymes tested alone. Incubation with a mixture of four enzymes was the only treatment which approximated the senescent process in the leaf, causing 34% loss of chlorophyll at pH 5 and 40% loss of protein at pH 7.4, both in 72 hours.In white light, the chlorophyll and the carotenoids, but not the protein, disappear rapidly. This disappearance was shown to be prevented in an atmosphere of nitrogen or in air by a number of reducing agents, of which ascorbic acid was the most effective. It is, therefore, ascribed to photooxidation rather than to normal senescence.     

Senescence of Rice Leaves XXX Levels of Endogenous Polyamines and Dark-Induced Senescence of Rice Leaves

The role of endogenous polyamines in the control of dark-inducedsenescence of detached rice leaves was investigated by quantitatinglevels of various polyamines by HPLC. Putrescine, spermidineand spermine were all present throughout senescence. Neithercadaverine nor 1,3-diaminopropane was detected. During dark-inducedsenescence, there was a marked decrease in levels of putrescineand an increase in those of spermidine and spermine. The rateof production of ethylene increased markedly upon excision ofleaves. -Difluoromethylarginine (DFMA) and -difluoromethylornithine(DFMO) caused a reduction in levels of putrescine, yet had noeffect on levels of spermidine and spermine. Neither DFMA norDFMO had any effect on senescence or on the production of ethylene.Treatment with dicyclohexylamine (DCH) and methylglyoxal bis-(guanylhydrazone)(MGBG) reduced levels of spermine and increased those of putrescinein detached leaves. After treatment with DCH or MGBG, both senescenceand the production of ethylene were significantly promoted.The current results suggest that endogenous polyamines may notplay a significant role in the control of dark-induced senescenceof rice leaves. This conclusion is supported by the furtherobservations that (a) benzyladenine, which is known to retardsenescence, decreased levels of putrescine but had no effecton those of spermidine and spermine; and (b) ABA, which promotedsenescence, increased levels of putrescine and had no effecton those of spermidine and spermine. (Received March 30, 1991; Accepted June 27, 1991)     

Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

Effects of compounds that influenced calcium uptake and calmodulininhibitors on the senescence of detached rice leaves were examined.Chelators, ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraaceticacid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraaceticacid (BAPTA), significantly promoted senescence of detachedrice leaves in the dark and light. The effect of EGTA can bereversed by treating detached rice leaves with calcium. Verapamil,a calcium channel blocker, and lanthanum chloride, a calciumantagonist, promoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. Calcium ionophore A23187 [GenBank] and ruthenium red, believed to raise cytosolic level of Ca²⁺,were quite effective in retarding dark-induced and ABA-promotedsenescence of detached rice leaves. Calmodulin inhibitors, W-7,compound 48/80, chlorpromazine and trifluoperazine, significantlypromoted dark-induced, and suppressed BA- and light-retardedsenescence of detached rice leaves. It is concluded that cytosoliclevel of Ca²⁺ may regulate senescence of detached rice leavesthrough a calmodulin-dependent mechanism. (Received June 13, 1990; Accepted August 3, 1990)