Journal of Fruit and Ornamental Plant Research Vol. 19(2) 2011: 123-131 FLOWER DEVELOPMENT AND SENESCENCE IN Ranunculus asiaticus L. Waseem Shahri and Inayatullah Tahir Department of Botany, University of Kashmir, Srinagar- 190006, INDIA Running title: Petal Senescence e-mail: [email protected] (Received November 24, 2010/Accepted July 7, 2011) ABSTRACT Flower development of Ranunculus asiaticus L. growing in the University Bo- tanic Garden was divided into six stages (I – VI): tight bud stage (I), loose bud stage (II), half open stage (III), open flower stage (IV), partially senescent stage (V) and senescent stage (VI). The average life span of an individual flower after it is fully open is about 5 days. Membrane permeability of sepal tissues estimated as electrical conductivity of ion leachates (µS), increased as the development proceeded through various stages. The content of sugars in the petal tissues increased during the flower opening period and then declined during senescence. The soluble proteins registered a consistent decrease with the simultaneous increase in specific protease activity and α-amino acid content during different stages of flower development and senescence. The content of total phenols registered an initial increase as the flowers opened, and then declined during senescence. Keywords: α-amino acids, flower senescence, membrane permeability, protease ac- tivity, soluble proteins, Ranunculus asiaticus, tissue constituents INTRODUCTION in gene expression and requires ac- tive gene transcription and protein Senescence comprises those proc- translation (Yamada et al., 2003; esses that follow physiological matur- Hoeberichts et al., 2005; Jones, ity leading to the death of a whole 2008). Flower petals are ideal tissues plant, organ or tissue, at the macro- for cell death studies as they are scopic level as well as microscopic short lived. Flower tissue is relatively level. It is a dynamic, closely regu- homogenous, thus chemical manipu- lated developmental process which lation can be applied without sub- involves highly coordinated changes stantial wounding. Petal senescence W. Shahri and I. Tahir has been found to be accompanied by as the Persian butter cup. The plants increase in the activity of catabolic have bowl-shaped or cup to saucer enzymes, ion leakage, and nuclear shaped flowers with a range of beau- fragmentation. This is all directed tiful colours, borne singly or in towards mobilization of nutrients cyme-like panicles. The plant re- from petals to other parts of the plant quires a mild climate. Flowering e.g. developing ovary (Halevy and occurs during spring and early sum- Mayak, 1979; Xu and Hanson, 2000; mer in outdoor gardens or during the Zhou et al., 2005; Chapin and Jones, winter and spring in greenhouses 2007; van Doorn and Woltering, (Fig. 1). 2008). Ethylene has been shown to modulate senescence in a number of MATERIAL AND METHODS flowers (Woltering and van Doorn, 1988; van Doorn, 2001; Shahri and Flowers of Ranunculus asiaticus Tahir, 2011a). The present investiga- growing in the Kashmir University tion has been undertaken on Ranun- Botanic Garden were used. Flower culus asiaticus to understand the development and senescence was changes occurring during flower divided into six stages. These stages development and senescence, with were deciphered as the: tight bud the aim to improve the postharvest stage (I), loose bud stage (II), half performance of this flower. It is an open stage (III), open flower stage ethylene-insensitive perennial geo- (IV), partially senescent stage (V) phyte that has a tuberous root and and senescent stage (VI). Visible segmented leaves characteristic of changes were recorded throughout the family Ranunculaceae (Kenza flower development and senescence et al., 2000). It is commonly known (Fig. 3 a). Figure 1. Flowers of Ranunculus asiaticus in full bloom 124 J. Fruit Ornam. Plant Res. vol. 19(2) 2011: 123-131 Flower development and senescence in Ranunculus asiaticus L. Figure 3. Stages of flower development and senescence in Ranunculus asiaticus (a) and increase in the pistil dimensions during various stages of flower development and senescence (b) Floral diameter, fresh and dry Total soluble sugars were estimated mass of 10 flowers as well as pistils after enzymatic conversion of non- were determined at each stage. Dry reducing sugars into reducing sugars mass was determined by drying the with invertase (BDH). Non-reducing material in an oven at 70 oC for 48 h. sugars were calculated as the differ- Changes in membrane permeability ence between total and reducing sug- were estimated by measuring the elec- ars. α-amino acids were estimated trical conductivity (µS) of leachates of (Rosen method, 1957) using glycine 5 petal discs per flower (5 mm in di- as the standard. Total phenols were ameter) punched from outer regions of estimated by the method of Swain petals of five different flowers incu- and Hillis (1959) using gallic acid as bated in a 15 ml glass of distilled water the standard. for 15 h at 20 oC. Proteins were extracted from 1 g At each stage, 1 g of chopped of petal tissue drawn separately from material of the petal tissue drawn 5 different flowers. The tissue was from 5 different flowers was fixed in homogenized in 5 ml of 5% sodium hot 80% ethanol. The material was sulphite (w/v) adding 0.1 g of poly- macerated and centrifuged three vinylpyrrolidone (PVP), and centri- times. The supernatants were pooled fuged. Proteins were precipitated and used for the measurement of the from a suitable volume of the cleared amount of sugars, amino acids and supernatant with an equal volume of total phenols. Reducing sugars were 20% trichloroacetic acid (TCA), determined by the method of Nelson centrifuged at 1000 x g for 15 min- (1944) using glucose as the standard. utes. The pellet was redissolved in J. Fruit Ornam. Plant Res. vol. 19(2) 2011: 123-131 125 W. Shahri and I. Tahir 0.1 N NaOH. Proteins were esti- RESULTS mated from a suitable aliquot by the method of Lowry et al. (1951) using The greenish buds open into bril- Bovine serum albumin (BSA) as the liant red flowers with a cluster of standard. brownish stamens at the centre sur- For protease activity determina- rounding the pistil. The stamens turn tion, at each stage 1 g pre-chilled blackish and abscise at senescent petal tissue (in 5 replicates) was ho- stage, whereas the petals showed mogenized in 15 ml chilled 0.1 M a typical colour change from dark red phosphate buffer (pH 6.5) in a pre- to brick red. The petals loose shape, cooled glass pestle and mortar. The become flaccid and begin to wither. contents were squeezed through four The withered petals finally abscise layers of muslin cloth and centri- upon slight teasing and the pistils fuged for 15 minutes at 5000 x g in increase in dimensions and develop a (Remi K- 24) refrigerated centri- into fruit (etaerio of achenes). The fuge at -5 oC. The supernatant was average life span of an individual used for the assay of protease activity flower after it opens fully is about by the method of Tayyab and Qamar 5 days. Diameter, fresh mass, dry (1992), with modification. The reac- mass and water content of flowers tion mixture comprised 1 ml of 0.1% increased as the flower development BSA dissolved in a 0.1 M phosphate progressed up to stage IV, and then buffer (pH 6.5). The reaction was declined as the senescence pro- stopped by adding 2 ml of 20% cold gressed through stages V and VI TCA. Blanks in which TCA was (Fig. 2a). Membrane permeability added prior to the addition of the estimated as electrical conductivity enzyme extract were run along with of ion leachates (µS) from petal each sample. The contents were cen- discs, registered a gradual increase trifuged and supernatants collected. during various stages of flower de- Free amino acids were estimated (as velopment and senescence (Fig. 2b). tyrosine equivalents) in a suitable Throughout flower development and aliquot of the supernatant by the senescence, the fresh and dry mass of method of Lowry et al. (1951), using the pistil increased, however, the tyrosine as the standard. The specific increment of increase was pro- enzyme activity was expressed as µg nounced during stages IV to VI (Fig. tyrosine equivalents liberated per mg 3b and 2c). of protein in the tissue extract. The tissue content of total and Each value represented in the ta- reducing sugars increased through bles corresponds to the mean ± S.E stages I to V and then quickly de- of five to ten independent replicates. clined during senescence (VI). The The data has been analyzed statisti- concentration of non-reducing sugars cally and LSD computed at P0.05 us- initially increased through stages I to ing MINITAB (v 15. 1.2- III, decreased during stages IV and V EQUINOX_Softddl.net) software. but registered a slight increase at 126 J. Fruit Ornam. Plant Res. vol. 19(2) 2011: 123-131 Flower development and senescence in Ranunculus asiaticus L. a) changes in fresh and dry mass of flowers b) changes in conductivity of ion leachates d) changes in the content of sugar fractions c) changes in fresh and dry mass of pistils e) changes in the content of soluble proteins f) changes in the content of á-amino acids and and specific protease activity total phenols Figure 2. Changes of same physiological parameters during various stages of flower development and senescence. Vertical bars with their corresponding markers represent LSD at P0.05 stage VI. The concentration of non- concentration of soluble proteins reducing sugars was much lower registered an increase as the devel- compared to that of reducing sugars opment progressed from stage I to II during stages III to VI (Fig.