Biochem. Physio!. Pflanzen 177. 275- 277 (1982)

Short Communication

Retarding Effect of Distilled Water Infiltration on Chlorophyll Breakdownl )

KAMLESH R. PATEL') and Y. D. SINGH3)

Key Term Index: senescence, water infiltration, chlorophyll breakdown; Datura stramonium

Summary

ChlorophyUlevel in leaf discs of Datura stramonium declined sharply after excision, whilst discs ,hich were infiltrated with distilled water maintained their chlorophyll content at the initial level ven after 10 d of incubation. Etluel, a potent promoter of senescence, had no effect on the chloro­,hyillevel of the discs infiltrated with distilled water. However, the protein content of control as well s infiltrated leaf discs decHned at about the same rate. It is concluded that chlorophyll degradation, widely accepted parameter offoliar senescence, may not be an inevita.ble part of aging phenomenon, nd chlorophyll and protein breakdown Me not inter·related processes.

Foliar senescence is characterised by a decline in the levels of starch, proteins, nucleic cids and especially the chlorophyll pigments. Excised leaves senesce rapidly, the loss f protein and subsequent disruption of chloroplasts result in yellowing of green leaves jHlBNALL 1939). Based on such observations, chlorophyll content has been considered s an indicator of senescence. Its retention in aging leaves is also employed in bioassays Ir growth regulators like gibberellic acids and cytokinins (KENDE 1964; WHYTE and ,UCKWILL 1966). Recently many growth h,hibitors (KNYPL 1970) and inhibitors of :NA and protein synthesis (KNYPL and MAZURCZYK 1972) have also been shown to Jtard the chlorophyll and protein breakdown in senescing leaves. It is generally believed lat chlorophyll and protein breakdown are inter-related and occur almost simultane­Isly in the senescing leaves (LEopom 1975; SPENCER and TITUS 1972; WOOLHOUSE )78). However, the present report indicates that under certain conditions the leaves tain their green color but senescence may set in as indicated by protein breakdown.

Healthy, mature leaves of uniform size and age of Datura stramonium, growing in the botanical xden of Gujarat· Uniyersity, Ahmedabad, India, were used in these experiments. The leaves were nace sterilized with 70 % ethanol for 30 s and immediately washed with sterile distilled water. laf discs were punched with a cork borer (15 mm diameter), avoiding main vein areas. The discs )rn one half of a leaf were completely infiltrated with sterile distilled wl.~ter using suction pump. lese infiltrated discs were kept in Petri dishes over filter papers moistened with 6 Illi of distilled

1) A part of Ph. D. thesis of K. R. PATEL, submitted to the Guj:uat University, Ahmedabad,

dia. 2) Greeley Memorial L~boratory! School of Forestry and Environmental studies, Yale Univec·

y, New Haven, U.S.A. 3) Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India.

276 K. It PATEL and Y. D. SINGH

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Fig. 1. Changes of chlorophyll levels. Control I .. ! discs (e- - e ); discs infiltrated with distilled water CA.--. ); infiltrated discs incuba.ted in the presence of ethrel (. 11).

Fig. 2. Chatlges of protein COtltenis. Control leaf discs (e--e); discs infiltrated with distilled water (.--.).

water or 100 ppm ethrel. The uninfiltrated leaf discs from the other half of the leaf which were kept on filter paper moistened with distilled water, served as the control. These Petri dishes were incubated in a dark room at 25 ± 2 °0. At intervals of 2 d, discs were removed, blotted dry and analysed for chlorophyll and protein contents. The chlorophyll was extracted with 80 % acetone and measured on Beckma.n DU-2 spectrophotometer at 660 nm. The protein content was determined by Lowry's method (Lowny et al. 1951). The {'xperiments were carried out in triplicates, each consisting of 5 discs.

The chlorophyll content of leaf discs infiltrated with distilled water do not change significantly during the entire period of incubation (Fig. 1). The infiltrated discs floated on ethrel also retain their chlorophyll nearly at presenescence level, but in control leaf discs the chlorophyll content declined sharply (Fig. 1). Surprisingly the protein content of control and infiltrated discs showed a parallel decline as the incubation period ad­vanced (Fig. 2). These rcsults lead to the conclusion that chlorophyll levels, under certain conditions , cannot be regarded as an indicator of senescence and the classical conclusion of CHIBNALL (1939) may not hold good in all cases. It is possible, as observed in our case, that the leaves may retain the green color and yet senescence may set in as evidenced by protein breakdown. Our contention is supported by work of THOMAS (1974) who showed that prolonged treatment of excised leaf sections with cycloheximido caused considerable damage to enzyme system in the tissue but at the same time main­tained cillorophyll at the pre-senescence level. Further support comes from the work of THOMAS and STODDART (1975) on a mutant of Festuea pratensis which does not cata­bolize chlorophyll in senescent leaves although protein breakdown and other aging processes take place. They also observed that abscisic acid promoted pigment loss in the wild type of Festuea pratensis but had no effect on chlorophyll in the mutant genotype. We also did not observe any change in the chlorophyll level in the infiltrated leaf discs

Water Infiltration and Chlorophyll Breakdown 277

floated on ethrel, a potent senescence promoter. From their studies on senescence in aquatic angiosperms, JANA and CHOUDHURY (1980) concluded that there is no parallel. ism in the degradation of chlorophyll and protein with age in Hydrilla, as is generally observed in terrestrial plants.

This simple experiment cannot explain the complexity of chlorophyll retention in lpite of protein breakdown. It is possible that distilled water infiltration alters the perme­ability of the tonoplast and the released enzymes degrade the structural and other en­zymic proteins including chlorophyllase, which occurs as an event of genetically pro­grammed senescence (THOMAS and STODDART 1980). However, it can be concluded that chlorophyll degradation, an universally accepted parameter of foliar senescence may not be an inevitable part of aging phenomenon and chlorophyll and protein breakdown lre not inter-related processes.

KAMLESH R. PATEL is thanktul to the Ministry of Education, Government of India, for the lward of national post doctoral fellowship.

References

DHIBNALL, A. C.: Protein metabolism in the plant. Yale Univers ity Press, New Haven 1939. JAN A, B. S., and CnOUDIiURY, M. A.: Senescence in submerged aquatic angiosperms: changes in

intact and isolated leaves during aging. New Phytol. 86, 191- 198 (1980). K EN DE, H.: Preservation of chlorophyll in leaf sections by substances obtained from root exudate.

Science 141i, 1066- 1067 (1964). KNYPL, J. S. : Arrest of yellow ing in senescing leaf discs of maize by growth retardants, coumarin and

inhibitors of RNA and protein synthesis. BioI. Plant. 12, 199- 207 (1970). - and MAZURCYZK, W.: Retarding effect of inhibitors of protein and RNA synthesis on chlorophyll

<lnd protein breakdown. BioI. Plant. 14, 146- 154 (1972). L EO POLD, A. C.: Aging, senescence a.nd turnover in plants. Bioscience 25, 659-662 (1975). LOWRY, O. H., ROSE UROUG II , N. J., FARH , A. L., and RANDALL, R. J.: Protein measurement with the

foIin reagent. J. BioI. Chern . 193, 265- 275 (1951). 31'ENCER, P. W., and TIT US , J. S.: Biochemical and enzymatic changes in apple leaf tissue during

autumnal senescence. lliant Physiol. 49 , 746- 750 (1972) . THOMAS, H.: Regulation of alanine aminotransferase in leaves of Lolium tomulen lum during senes­

cence. Z. P!lanzenphysiol. 74, 208- 218 (1974). - and STODD :\RT, J. L.: Separation of chlorophyll degradation from other senescence processes in

leaves of a mutant genotype of meadow fescue (Festuca pratensis). Plant Physiol. 66, 438-441 (1975).

- - Leaf senescence. Ann. Rev. Plant Physiol. 31 , 83-111 (1980). WHYT E, P., and LUCKWILL, L. C.: Sensitive bioassays for gibberellins based on retardation of leaf

senescence in Rumex obtusl:{olium. Nature 210, 1360 (19G6). WOOI.IIO US E, H. \Y.: Ce llular and metabol ic aspects of senescence in higher pla.nts. In: The Biology of

Aging (Edit. J. A. llENIIKE, C. E. FINCII, G. B. MOMENT), pp. 83-89 . New York: Plenum 1978.

Recei !:cd July 26; in revised form October 10, 1981

Authors' addresses: KAMLESII R. PATEL, Gree ley Memorial Laboratory, School of Forestry and Environmental studies, Ya le University, New Haven, C. T. 06511 U.S.A.; Y. D. SINGH, Department )f Bioscicnces, Saurashtra University, Rajkot, Gujarat. India.