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Carbohydrate Changes in Shoot Tip and Subtending Leaves During Ontogenetic Development of Pineapple
Carbohydrate Changes in Shoot Tip and Subtending Leaves During Ontogenetic Development of Pineapple K. N. MADHUSUDANAN and S. NANDAKUMAR Department of Botany, University of Calicut, Calicut University, Kerala - 673 635, India Received October 30,1982 . Accepted March 28,1983
Summary Starch and ethanol-soluble sugar components were determined in the shoot tip and in the achlorophyllous - and chlorophyllous part of the subtending leaves during the ontogeny of the pineapple plant. The changes observed were correlated with rooting process in the propagule, vegetative growth of the plant and the change to the reproductive state inflorescence formation, induced by natural environmental conditions. The carbohydrate distribution pattern in the chlorophyllous leaf part was suggestive of dependence on the metabolic changes in the pineapple shoot apex. A physiological role for the achlorophyllous leaf part has been proposed. The significance of the distribution of carbohydrate between the readily assimilable soluble form (sugars) and the insoluble storage form (starch) has been briefly considered in relation to the tissue and the stage of growth.
Key words: Ananas comosus (L.) Merr., shoot tip, achlorophyllous and chlorophyllous leaf parts, induction.
Introduction
Carbohydrates are intimately associated with the process of flower initiation (Bernier, 1969; Evans, 1971, 1976; Vince-Prue, 1975). A correlation has been sought between the content and type of carbohydrates in the shoot apex and flowering in the higher plants. The earlier studies in this field and their limitations were reviewed by Schwabe (1971). Bodson (1977) reported that an increase in soluble sugar content was the earliest biochemical change detected in the «evoked» long-day Sinapis apex. (The events which occur at the shoot apex in the period between the arrival of the floral stimulus (from the leaf) and the first morphologically distinct signs of flowering have been collectively termed by Evans (1971) as evocation. The change from one particular morphological state to another, as for example, the change from the vegetative to the reproductive apex, is generally termed as transition.} The soluble sugar content increased also in the leaf at the time of induction (Bodson, 1977). An increase in starch concentration was also observed in the leaves and shoot tip of the induced plant (Kinet, 1975; Bodson, 1977), but this change was concluded to be unspecific and not immediately related to flowering. Flowering in the pineapple, a short-day plant (Gowing, 1961), has been the subject of investigation by a number of workers, but with the exception of the study by
z. Pjlanzenphysiol. Ed. 110. S. 429--438.1983.
430
K.
N. MADHUSUDANAN
and S. NANDAKUMAR
Kerns et al. (1936), in which the plants flowered under natural environmental conditions, the more critical studies were on plants forced to flower by application of exogenous chemicals (Gifford, 1969; Bartholomew and Kadzimin, 1977). These studies related to the morphological, anatomical and histological and histochemical features of flowering, but the biochemical reactions were not investigated. The pineapple plant is probably unique in that it can be forced to flower at any stage of growth by the external application of growth factors (Collins, 1968). Madhusudanan et al. (1983) observed isolated instances of juvenile pineapple plants putting out inflorescence under natural environmental conditions. A complete study of flowering in pineapple should, therefore, include the ontogenetic development of the shoot apex. The following is a study of the metabolizable carbohydrates in the shoot tip of Kew cultivar of pineapple, from the stage of propagule planting to the stage of inflorescence formation under natural environmental conditions and the incipient reversion of the inflorescence apex to the vegetative state. Simultaneously with the shoot tip, the subtending leaves were analysed for carbohydrate constituents, separately on the achlorophyllous- and chlorophyllous part.
Materials and Methods Plant material. Ananas comosus (L.) Merr., Kew cultivar, was raised from hapas (the propagules which develop at the junction of the peduncle with the main stem) as described by Madhusudanan et al. (1983). Stages o/sample collection. The first stage was the hapa, ready for planting. Subsequent collections were at 2-month intervals (stages 2-8) up to 14 months, when the plant was apparently still in the vegetative state. Stage 9, identified, with the help of field lens by the formation of the first kinked bract and stage 10, identified by visible doming and clustering of bristle-like bracts at the summit, corresponded respectively to the transitional (evocative) - and organogenetic stage. Stage 11 was when all florets differentiated on the inflorescence, but anthesis occurred in none and incipient crown formation took place. Tissues analysed Shoot tip. T e defoliated shoot tips were excised 5 mm below the summit in stages 1 to 10. In stage 11, the entire inflorescence was collected and bracts cut away. Two to three samples were collected at a time, cut into small pieces and portions taken for analyses. Leaves. The top-most leaves, which were totally nonchlorophyllous, numbering 10-12, were discarded. The next set of leaves, numbering 10-12, inclusive of the «D» leaves, were used for analyses. The achlorophyllous and chlorophyllous leaf parts were excised and the intervening semichlorophyllous region discarded. The two parts were separately cut into small bits and samples used for analyses. Sample collections at every stage were at 8 a.m. The shoot tips and leaf parts at the various stages and inflorescence in stage 11 were analysed a minimum of five times and the data evaluated statistically. Analyses The analyses were carried out on the fresh tissues. The results were expressed in terms of unit dry weight of the tissues, since the moisture content varied with the tissue and with the stage of
Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
431
growth. Maceration of the fresh tissues was in a chilled glass mortar with pestle, using sand as abrasive. Starch. Starch was precipitated from 200 mg tissue according to Whelan (1955). The starch pellet was hydrolysed with 10 % (v/v) H2S04 and aliquots used for carbohydrate determination according to Montgomery (1957), using soluble starch as standard. Ethanol-soluble sugars. Sugars were extracted from 2 g tissue with hot 80 % (v/v) ethanol and the solvent-freed preparation purified by passing through a column of Dowex 50-x8. Sugar components were separated by descending paper chromatography, employing the solvent system n-butanol : acetic acid : water (4: 1 : 5) and run in duplicate for 80 h. The spots were visualised in one of the chromatograms by spraying with diphenylamine-anilineorthophosphoric acid reagent and identified by comparing with a simultaneously prepared chromatogram of a known mixture. By superimposing the sprayed on the unsprayed chromatogram, the sugars were marked off and were then eluted with hot water. Estimation of eluted sugars was according to Montgomery (1957), using as standards the particular sugars eluted from (the unstained) standard chromatogram.
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Fig. 1: Starch and ethanol-soluble sugars in the shoot tip of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose ( -0 -); Glucose ( -6 -); Fructose
(-e-).
Z. Pjlanzenphysiol. Ed. 110. S. 429-438. 1983.
432
K. N. MADHUSUDANAN and S. NANDAKUMAR
Results Figures 1-3 illustrate the changes in starch and glucose, fructose and sucrose; the data for the total metabolizable carbohydrate and its distribution between starch and sugars are recorded in Table 1. Starch. At any stage, starch concentration in shoot tip far exceeded that in the chlorophyllous - or achlorophyllous leaf part; at all stages of vegetative growth (stages 1-8), except stage 2, achlorophyllous leaf part had a markedly higher starch concentration than the chlorophyllous part. The hapa had a high concentration of starch in all tissues analyzed. At stage 2, there was a considerable drop in starch in the tissues. In the early phase of the vegetative growth (stages 3-5), starch concentration was restored in the shoot tip and the achlorophyllous and chlorophyllous leaf parts. Commencement and progress of the accelerated phase of stem growth (a form of «stem bolting»), during the latter half of 100
25
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Fig. 2: Starch and ethanol-soluble sugars in the achlorophyllous leaf part of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose (-0-); Glucose (-/:;.-); Fructose (-e-).
z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
433
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Fig. 3: Starch and ethanol-soluble sugars in the chlorophyllous leaf part of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose ( -0 -); Glucose ( -6 -); Fructose (-e-).
the vegetative state, was associated with a decreased level of starch in all parts, but the decrease was least in the shoot tip. There was a marked decrease in starch concentration in the shoot tip in the prefloral (stage 9) and floral stage (stage 10). Starch decreased also in the achlorophyllous leaf part in the prefloral stage, but it increased in the chlorophyllous leaf part; in both, an increase ensued in the floral stage. In stage 11, there was a marked increase in starch concentration in the inflorescence and in the achlorophyllous and chlorophyllous leaf parts. Separate studies in which the ethanol-insoluble residue from tissues of the hapa (extracted in the presence of added CaC03) was extracted with hot water and the extract precipitated with ethanol and the precipitate tested with resorcinol, showed that inulin was absent. Other studies showed that the ethanol-soluble form of fructosan was also absent. Ethanol-soluble sugars. The major sugars in the three tissues were sucrose, glucose and fructose; maltose and raffinose occurred in lower concentrations. Glucose and fructose occurred in the shoot tip in equal amounts in most of the stages. The two Z. Pjlanzenphysiol. Bd. 110. S. 429--438. 1983.
434
K.
N. MADHUSUDANAN
and S.
NANDAKUMAR
Table 1: Total metabolizable carbohydrate and its distribution between starch and sugars in the shoot tip and subtending leaves of Ananas comosus during vegetative and reproductive growth. Tissue
Stage of development
3
4
5
7
6
10
11
108
68
117
33
47
38
86
10
25
18
57
9
1
2
8
299
79
297
311
264
186
220
197
100
29
77
72
82
134
30
38
18
37
22
40
41
13
Total metabolizable carbohydrate I), mg/g dry wt. Shoot tip Achlorophyllous leaf Chlorophyllous leaf
Ratio starch: ethanol-soluble sugar2) Shoot tip Achlorophyllous leaf Chlorophyllous leaf
7.8
7.8
30
47
24
14
21
2.2
0.4
2.7
4.5
1.4
4.0
1.6
2.4
0.8
2.9
2.2
2.1
1.2
0.6
37
2.5
2.0
5.7
0.4
0.1
0.3
0.4
0.7
0.4
0.8
1.2
I. 2) For calculating total metabolizable carbohydrate and the ratio starch to sugar, the polysaccharide, oligosaccharide and the disaccharides were converted to their hexose equivalents by the use of appropriate factors.
hexoses occurred in nearly equal quantities also in the achlorophyllous leaf part, but fructose exceeded glucose in the chlorophyllous leaf part in many stages. Galactose appeared in trace quantities in a few stages. In the hapa, sucrose concentration was high in the shoot tip and in the achlorophyllous leaf part. Sucrose concentration was low in the chlorophyllous leaf part of the hapa, but this was higher than at any other stage of vegetative growth. Following planting, a marked reduction occurred in sucrose, glucose and fructose concentrations in the shoot tip (stage 2); they were never restored to the original high concentration in any of the subsequent vegetative stages. Glucose was at its lowest concentration, and fructose in traces, in the final vegetative stage. Considerable sucrose reduction occurred also in the achlorophyllous and chlorophyllous leaf parts following planting of hapa. Whereas sucrose in the achlorophyllous leaf part fluctuated in concentration in the subsequent stages of vegetative growth, it generally remained at a low level in the chlorophyllous leaf part. Glucose and fructose were in high concentration in the achlorophyllous leaf part following planting and this high concentration was more or less maintained during the rest of vegetative phase. There was a wide fluctuation in glucose and fructose concentration in the chlorophyllous leaf part throughout the vegetative stage of growth, but fructose in particular was maintained at a high level. During evocation (stage 9), sucrose concentration in the shoot tip increased 7-fold; glucose concentration was 3-fold higher and fructose was now present in assayable Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
435
amounts. There was a doubling in sucrose concentration in the achlorophyllous leaf part and over 8-fold increase in the chlorophyllous leaf part. Glucose and fructose at this stage showed a 2-fold increase in the achlorophyllous and chlorophyllous leaf parts. With morphogenesis at the shoot apex, sucrose underwent a marked decrease in the concentration in the shoot tip, while glucose and fructose increased. Sucrose and the two hexoses decreased in the achlorophyllous and chlorophyllous leaf parts. In stage 11, there was an increase in the sucrose concentration in the early inflorescence and in the leaf parts. Glucose level decreased and fructose was reduced to trace concentration in the shoot tip, but there was a marked increase in glucose and fructose in the achlorophyllous and chlorophyllous leaf parts. Maltose was a quantitatively minor component of the shoot tip in all stages (0.4 to 2.9 mgg- I ); it was reduced to trace concentration during evocation. In the penultimate stage of vegetative growth (stage 7), maltose could not be detected in the achlorophyllous leaf parts; in all the other stages, maltose was present (0.7 to 3.5mgg- 1 in the achlorophyllous and 0.5 to 2.6mgg- 1 in the chlorophyllous leaf part). When the shoot apex was undergoing evocation reactions, maltose decreased to trace concentration also in the achlorophyllous leaf part, but it was present in 1 mg g-I concentration in the chlorophyllous leaf part. Raffinose occurred in the tissues of the pineapple plant in many stages, in concentration comparable with that of maltose, (0.4 to 1.5mgg- 1 in the shoot tip, 0.7 to 2.1 mgg- I in the achlorophyllous leaf part and 0.4 to 1 mgg- I in the chlorophyllous leaf part). In stage 2, however, raffinose was absent in all tissues. During evocation, raffinose was in trace concentration in the shoot tip and achlorophyllous leaf part, but it was present in appreciable concentration (0.8 mg g-I) in the chlorophyllous leaf part. Starch in the shoot tip was at all stages more abundant than total ethanol-soluble sugars, both expressed as hexose equivalent. The ratio starch: ethanol-soluble carbohydrate was in intermediate value in the hapa stage and was unaltered following planting. This ratio was maintained at a fairly high level throughout the rest of the vegetative state. At the time of evocation, the ratio abruptly decreased in the shoot tip. The ratio remained low also during morphogenesis, but increased in the early stage of inflorescence. In the achlorophyllous leaf part, starch exceeded ethanol-soluble carbohydrate in the vegetative state, except following planting of hapa and in the last stage. The ratio starch: total ethanol-soluble carbohydrate was always lower in the achlorophyllous leaf part than in the shoot tip. Marked reduction in the ratio was evident following planting and during evocation. The chlorophyllous leaf part generally resembled the achlorophyllous leaf part in the pattern of distribution of total ethanol-soluble sugars, total metabolisable carbohydrate and the ratio starch: total ethanol-soluble sugars.
z. Pjlanzenphysiol. Ed. 110. S. 429-438. 1983.
436
K. N. MADHUSUDANAN and S. NANDAKUMAR
Discussion The high concentration in which starch and soluble sugars occurred in the tissues of the hapa was, probably, to be traced to the dual means of carbohydrate nutrition, supply from the mother plant and photosynthesis by propagule. The constancy in the ratio starch: ethanol-soluble carbohydrate in stage 2 in the shoot tip and its decrease in the leaf parts suggested that the partition between starch and sugars had to be maintained constant to meet the physiological needs of the shoot tip, in these stages, but that in the leaf the emphasis was on the readily metabolizable and transportable form rather than the storage form. The marked decrease in starch and sugar content in the leaf parts and particularly in the shoot tip observed in stage 2 reflected the stress the propagule was subjected to when forced to grow as an autonomous plant. It also pointed to the biochemical basis for the adverse effect of undue storage of propagules in plant crop growth, reported by Norman (1980), since excessive loss of reserves and metabolites may not be reversed on subsequent planting. The precipitous decrease in starch concentration in the shoot tip in stage 2 was analogous to the events occurring in cotyledons/endosperm during germination and seedling development (Mayer and Poljakoff-Mayber, 1975). Throughout stages 3 to 7, there was no qualitative change in carbohydrate constituents, nor was an abrupt quantitative change evident at any stage. The absence of any abrupt changes was to be expected from the susceptibility of the plant to forced flowering at any stage, even though the fruit size depended on the growth stage at which the hormone was applied (Kerns et aI., 1936). The most significant changes in carbohydrate levels and distribution during ontogeny were those attendant on the transition of the vegetative to the reproductive state. In stage 8, the plant was fully mature and apparently receptive to the floral stimulus which was being elaborated in the interval between stages 7 and 8. Starch decreased insignificantly in shoot tip in stage 8 and markedly in stage 9, when evocation reactions were about to be initiated (stage 8), or had advanced (stage 9), irrevocably committing the apex to flowering. There was a spurt in sucrose level in shoot tip in stage 9, which was analogous to the increase in soluble sugars which Bodson (1977) observed in evoked Sinapis apex, but of much higher magnitude. Simultaneously, there was an increase in sucrose concentration in the achlorophyllous leaf part and more so in the chlorophyllous leaf part. Starch decreased in the achlorophyllous leaf part in stage 9 (P < 0.01), but increased in the chlorophyllous leaf part (P
z. Pjlanzenphysiol. Bd. 110. S. 429-438.1983.
Carbohydrate changes during development of pineapple
437
Glucose was at its lowest level in the shoot tip and fructose occurred in traces in stage 8; both increased in stage 9. In both chlorophyllous and achlorophyllous leaf part, the two hexoses increased in concentration in stage 9, either hexose being present in markedly higher concentration than sucrose. The primary purpose of glucose and fructose accumulation in the achlorophyllous leaf part in stages 8 and 9 seemed to be for transfer to the shoot tip, to meet its metabolic needs. The pattern of these changes would lead one to speculate that target cells for the flowering principle elaborated by the green leaf were present in the achlorophyllous leaf part in addition to the shoot apex. The ontogenetic pattern of distribution of carbohydrate components in the chlorophyllous leaf part suggested that the photosynthetic activity, or, at any rate, the distribution among the primary product (sucrose) and the derived products (glucose and fructose and starch) was dependent on the physiological stage of the plant, the shoot apex in particular. The pattern of maltose distribution in the chlorophyllous leaf part was difficult to reconcile with a role that it is merely a transient intermediate in starch degradation. Maltose can be biosynthesized in green leaves (Linden et aI., 1975). Its distribution pattern in the achlorophyllous leaf part and in the shoot tip was suggestive of an active physiological role for maltose in the metabolism of pineapple shoot apex, especially during evocative reactions when the sugar was reduced to trace level. To the authors' best of knowledge, this is the first time that evidence, though circumstantial, has been found for a distinctive metabolic role for maltose in a higher plant. That raffinose also played a role during the evocation reactions at the apex appeared likely from its presence in trace concentrations only in the shoot tip and achlorophyllous leaf part in stage 9. The ratio starch: ethanol-soluble carbohydrate during the developmental stages fell into a few groups. Stage 9 witnessed an abrupt lowering in this ratio in the shoot tip, suggesting an accentuation of the partition of carbohydrate in the form of readily metabolizable sugars, in preference to storage starch, at the time of floral evocation. In stage 11, when the apex of the early inflorescence axis was reverting to the vegetative state, the ratio was more than doubled, elevating it to nearly the same magnitude as in stages 1 and 2. Unlike in the shoot tip, the ratio in the achlorophyllous leaf part was frequently less than one. This was indicative of carbohydrates being held preferentially in a form readily translocated out of leaf. Total metabolizable (nonstructural) carbohydrate decreased markedly in the shoot tip during evocation (stage 9), but increased in the leaf. Acknowledgements The authors are grateful to Professor P. S. Krishnan, Emeritus Professor in Biochemistry, for his interest in the investigation and to Professor B. K. Nayar, Head of the Department, for laboratory facilities. Our gratitude is due to Professor Dr. R. Kollmann, Kiel, for helpful suggestions in the preparation of this manuscript. We are indebted to Professor C. C. Black, Georgia, at whose suggestion the test for fructosans was carried out.
z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
438
K. N. MADHUSUDANAN and S. NANDAKUMAR
References BARTHOLOMEW, D. P. and S. B. KADZIMIN: Pineapple. In: ALVIM, P. T. and T. T. KOZLOWSKI (Ed.): Ecophysiology of Tropical Crops, 113-156. Academic Press, Inc., New York, San Francisco, London, 1977. BERNIER, G.: Sinapis alba L. In: EVANS, L. T. (Ed.): The Induction of Flowering, 305-327. Macmillan of Australia, Melbourne, 1969. BODSON, M.: Changes in the carbohydrate content of the leaf and the apical bud of Sinapis during transition to flowering. Planta 135, 19-23 (1977). COLLlNS,J. L.: The Pineapple. pp. 295. Leonard Hill, London, 1968. EVANS, L. T.: Flower induction and the florigen concept. Annu. Rev. Plant Physiol. 22, 365-394 (1971). - Inhibition of flowering in Lolium temulentum by the photosynthetic inhibitor 3 (3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in relation to assimilate supply to the shoot apex. In: JACQUES, R. (Ed.): Etude de Biologie Vegetale. Hommage au Professuer P. Chouard, Paris, 1976. GIFFORD, E. M.: Initiation and early development of the inflorescence in pineapple (Ananas comosus «Smooth Cayenne») treated with acetylene. Amer. J. Bot. 48, 657-666 (1969). GOWING, D. P.: Experiments on the photoperiodic response in pineapple. Amer. J. Bot. 48, 16-21 (1961). KERNS, K. R., J. L. COLLINS, and H. KIM: Developmental studies of the pineapple Ananas com· osus (L.) Merr. 1. Origin and growth of leaves and inflorescence. New Phytol. 35, 305-317 (1936). KINET, J. M.: Gross chemical changes occurring in the leaf of Sinapis alba during photoperiodic induction of flowering. New Phytol. 74,25-32 (1975). LINDEN, J. c., N. SCHILLING, H. BRACKENHOFER, and O. KANDLER: Asymmetric labelling of maltose during photosynthesis in 14C02. Z. Pflanzenphysiol. 76, 176-181 (1975). MAoHUSUDANAN, K. N., E. NABEESA, V. UMADEVI, and S. NANDAKUMAR: Crop growth pattern and the propagule differentiation of 20 varieties in pineapple. Scientia Horticulturae 18, 215-224 (1983). MAYER, A. M. and A. POLJAKOFF-MAYER: The Germination of Seeds. pp. 192. Pergamon Press, Oxford, New York, Sec. Edn. 1975; reprinted, 1978. MONTGOMERY, R.: Determination of glycogen. Arch. Biochem. Biophys. 67, 378-386 (1957). NORMAN, J. c.: Effects of storage and type of planting material on pineapple, Ananas comosus cultivar sugarloaf. Gartenbauwissenschaft 45, 255-259 (1980). SCHWABE, W. W.: Physiology of vegetative reproduction and flowering. In: STEWARD, F. C. (Ed.): Plant Physiology, 6A, 233-411. Academic Press, New York and London, 1971. VINCE-PRUE, D.: Photoperiodism in Plants. pp. 444. McGraw-Hill, London, 1975.
Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Summary Starch and ethanol-soluble sugar components were determined in the shoot tip and in the achlorophyllous - and chlorophyllous part of the subtending leaves during the ontogeny of the pineapple plant. The changes observed were correlated with rooting process in the propagule, vegetative growth of the plant and the change to the reproductive state inflorescence formation, induced by natural environmental conditions. The carbohydrate distribution pattern in the chlorophyllous leaf part was suggestive of dependence on the metabolic changes in the pineapple shoot apex. A physiological role for the achlorophyllous leaf part has been proposed. The significance of the distribution of carbohydrate between the readily assimilable soluble form (sugars) and the insoluble storage form (starch) has been briefly considered in relation to the tissue and the stage of growth.
Key words: Ananas comosus (L.) Merr., shoot tip, achlorophyllous and chlorophyllous leaf parts, induction.
Introduction
Carbohydrates are intimately associated with the process of flower initiation (Bernier, 1969; Evans, 1971, 1976; Vince-Prue, 1975). A correlation has been sought between the content and type of carbohydrates in the shoot apex and flowering in the higher plants. The earlier studies in this field and their limitations were reviewed by Schwabe (1971). Bodson (1977) reported that an increase in soluble sugar content was the earliest biochemical change detected in the «evoked» long-day Sinapis apex. (The events which occur at the shoot apex in the period between the arrival of the floral stimulus (from the leaf) and the first morphologically distinct signs of flowering have been collectively termed by Evans (1971) as evocation. The change from one particular morphological state to another, as for example, the change from the vegetative to the reproductive apex, is generally termed as transition.} The soluble sugar content increased also in the leaf at the time of induction (Bodson, 1977). An increase in starch concentration was also observed in the leaves and shoot tip of the induced plant (Kinet, 1975; Bodson, 1977), but this change was concluded to be unspecific and not immediately related to flowering. Flowering in the pineapple, a short-day plant (Gowing, 1961), has been the subject of investigation by a number of workers, but with the exception of the study by
z. Pjlanzenphysiol. Ed. 110. S. 429--438.1983.
430
K.
N. MADHUSUDANAN
and S. NANDAKUMAR
Kerns et al. (1936), in which the plants flowered under natural environmental conditions, the more critical studies were on plants forced to flower by application of exogenous chemicals (Gifford, 1969; Bartholomew and Kadzimin, 1977). These studies related to the morphological, anatomical and histological and histochemical features of flowering, but the biochemical reactions were not investigated. The pineapple plant is probably unique in that it can be forced to flower at any stage of growth by the external application of growth factors (Collins, 1968). Madhusudanan et al. (1983) observed isolated instances of juvenile pineapple plants putting out inflorescence under natural environmental conditions. A complete study of flowering in pineapple should, therefore, include the ontogenetic development of the shoot apex. The following is a study of the metabolizable carbohydrates in the shoot tip of Kew cultivar of pineapple, from the stage of propagule planting to the stage of inflorescence formation under natural environmental conditions and the incipient reversion of the inflorescence apex to the vegetative state. Simultaneously with the shoot tip, the subtending leaves were analysed for carbohydrate constituents, separately on the achlorophyllous- and chlorophyllous part.
Materials and Methods Plant material. Ananas comosus (L.) Merr., Kew cultivar, was raised from hapas (the propagules which develop at the junction of the peduncle with the main stem) as described by Madhusudanan et al. (1983). Stages o/sample collection. The first stage was the hapa, ready for planting. Subsequent collections were at 2-month intervals (stages 2-8) up to 14 months, when the plant was apparently still in the vegetative state. Stage 9, identified, with the help of field lens by the formation of the first kinked bract and stage 10, identified by visible doming and clustering of bristle-like bracts at the summit, corresponded respectively to the transitional (evocative) - and organogenetic stage. Stage 11 was when all florets differentiated on the inflorescence, but anthesis occurred in none and incipient crown formation took place. Tissues analysed Shoot tip. T e defoliated shoot tips were excised 5 mm below the summit in stages 1 to 10. In stage 11, the entire inflorescence was collected and bracts cut away. Two to three samples were collected at a time, cut into small pieces and portions taken for analyses. Leaves. The top-most leaves, which were totally nonchlorophyllous, numbering 10-12, were discarded. The next set of leaves, numbering 10-12, inclusive of the «D» leaves, were used for analyses. The achlorophyllous and chlorophyllous leaf parts were excised and the intervening semichlorophyllous region discarded. The two parts were separately cut into small bits and samples used for analyses. Sample collections at every stage were at 8 a.m. The shoot tips and leaf parts at the various stages and inflorescence in stage 11 were analysed a minimum of five times and the data evaluated statistically. Analyses The analyses were carried out on the fresh tissues. The results were expressed in terms of unit dry weight of the tissues, since the moisture content varied with the tissue and with the stage of
Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
431
growth. Maceration of the fresh tissues was in a chilled glass mortar with pestle, using sand as abrasive. Starch. Starch was precipitated from 200 mg tissue according to Whelan (1955). The starch pellet was hydrolysed with 10 % (v/v) H2S04 and aliquots used for carbohydrate determination according to Montgomery (1957), using soluble starch as standard. Ethanol-soluble sugars. Sugars were extracted from 2 g tissue with hot 80 % (v/v) ethanol and the solvent-freed preparation purified by passing through a column of Dowex 50-x8. Sugar components were separated by descending paper chromatography, employing the solvent system n-butanol : acetic acid : water (4: 1 : 5) and run in duplicate for 80 h. The spots were visualised in one of the chromatograms by spraying with diphenylamine-anilineorthophosphoric acid reagent and identified by comparing with a simultaneously prepared chromatogram of a known mixture. By superimposing the sprayed on the unsprayed chromatogram, the sugars were marked off and were then eluted with hot water. Estimation of eluted sugars was according to Montgomery (1957), using as standards the particular sugars eluted from (the unstained) standard chromatogram.
300
~
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200 '" W 3:
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30
SHOOT TIP UJ
3: ~ 15
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g
10
V1
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--'
co
3o
,
V1
--' o
5
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I-
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STAGE OF DEVELOPMENT
Fig. 1: Starch and ethanol-soluble sugars in the shoot tip of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose ( -0 -); Glucose ( -6 -); Fructose
(-e-).
Z. Pjlanzenphysiol. Ed. 110. S. 429-438. 1983.
432
K. N. MADHUSUDANAN and S. NANDAKUMAR
Results Figures 1-3 illustrate the changes in starch and glucose, fructose and sucrose; the data for the total metabolizable carbohydrate and its distribution between starch and sugars are recorded in Table 1. Starch. At any stage, starch concentration in shoot tip far exceeded that in the chlorophyllous - or achlorophyllous leaf part; at all stages of vegetative growth (stages 1-8), except stage 2, achlorophyllous leaf part had a markedly higher starch concentration than the chlorophyllous part. The hapa had a high concentration of starch in all tissues analyzed. At stage 2, there was a considerable drop in starch in the tissues. In the early phase of the vegetative growth (stages 3-5), starch concentration was restored in the shoot tip and the achlorophyllous and chlorophyllous leaf parts. Commencement and progress of the accelerated phase of stem growth (a form of «stem bolting»), during the latter half of 100
25
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ACHLOROPHYLLOUS
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o:
""
Cl ::J
'"~
10
3 o
'", --'
o
z
..."" ::t:
5
UJ
"()
STAGE
11
OF DEVELOPMENT
Fig. 2: Starch and ethanol-soluble sugars in the achlorophyllous leaf part of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose (-0-); Glucose (-/:;.-); Fructose (-e-).
z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
433
]0
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15
10
E
:r.'"
u a:
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STAGE OF
DEVELOPMENT
Fig. 3: Starch and ethanol-soluble sugars in the chlorophyllous leaf part of Ananas comosus during vegetative and reproductive growth. Starch (-A-); Sucrose ( -0 -); Glucose ( -6 -); Fructose (-e-).
the vegetative state, was associated with a decreased level of starch in all parts, but the decrease was least in the shoot tip. There was a marked decrease in starch concentration in the shoot tip in the prefloral (stage 9) and floral stage (stage 10). Starch decreased also in the achlorophyllous leaf part in the prefloral stage, but it increased in the chlorophyllous leaf part; in both, an increase ensued in the floral stage. In stage 11, there was a marked increase in starch concentration in the inflorescence and in the achlorophyllous and chlorophyllous leaf parts. Separate studies in which the ethanol-insoluble residue from tissues of the hapa (extracted in the presence of added CaC03) was extracted with hot water and the extract precipitated with ethanol and the precipitate tested with resorcinol, showed that inulin was absent. Other studies showed that the ethanol-soluble form of fructosan was also absent. Ethanol-soluble sugars. The major sugars in the three tissues were sucrose, glucose and fructose; maltose and raffinose occurred in lower concentrations. Glucose and fructose occurred in the shoot tip in equal amounts in most of the stages. The two Z. Pjlanzenphysiol. Bd. 110. S. 429--438. 1983.
434
K.
N. MADHUSUDANAN
and S.
NANDAKUMAR
Table 1: Total metabolizable carbohydrate and its distribution between starch and sugars in the shoot tip and subtending leaves of Ananas comosus during vegetative and reproductive growth. Tissue
Stage of development
3
4
5
7
6
10
11
108
68
117
33
47
38
86
10
25
18
57
9
1
2
8
299
79
297
311
264
186
220
197
100
29
77
72
82
134
30
38
18
37
22
40
41
13
Total metabolizable carbohydrate I), mg/g dry wt. Shoot tip Achlorophyllous leaf Chlorophyllous leaf
Ratio starch: ethanol-soluble sugar2) Shoot tip Achlorophyllous leaf Chlorophyllous leaf
7.8
7.8
30
47
24
14
21
2.2
0.4
2.7
4.5
1.4
4.0
1.6
2.4
0.8
2.9
2.2
2.1
1.2
0.6
37
2.5
2.0
5.7
0.4
0.1
0.3
0.4
0.7
0.4
0.8
1.2
I. 2) For calculating total metabolizable carbohydrate and the ratio starch to sugar, the polysaccharide, oligosaccharide and the disaccharides were converted to their hexose equivalents by the use of appropriate factors.
hexoses occurred in nearly equal quantities also in the achlorophyllous leaf part, but fructose exceeded glucose in the chlorophyllous leaf part in many stages. Galactose appeared in trace quantities in a few stages. In the hapa, sucrose concentration was high in the shoot tip and in the achlorophyllous leaf part. Sucrose concentration was low in the chlorophyllous leaf part of the hapa, but this was higher than at any other stage of vegetative growth. Following planting, a marked reduction occurred in sucrose, glucose and fructose concentrations in the shoot tip (stage 2); they were never restored to the original high concentration in any of the subsequent vegetative stages. Glucose was at its lowest concentration, and fructose in traces, in the final vegetative stage. Considerable sucrose reduction occurred also in the achlorophyllous and chlorophyllous leaf parts following planting of hapa. Whereas sucrose in the achlorophyllous leaf part fluctuated in concentration in the subsequent stages of vegetative growth, it generally remained at a low level in the chlorophyllous leaf part. Glucose and fructose were in high concentration in the achlorophyllous leaf part following planting and this high concentration was more or less maintained during the rest of vegetative phase. There was a wide fluctuation in glucose and fructose concentration in the chlorophyllous leaf part throughout the vegetative stage of growth, but fructose in particular was maintained at a high level. During evocation (stage 9), sucrose concentration in the shoot tip increased 7-fold; glucose concentration was 3-fold higher and fructose was now present in assayable Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
Carbohydrate changes during development of pineapple
435
amounts. There was a doubling in sucrose concentration in the achlorophyllous leaf part and over 8-fold increase in the chlorophyllous leaf part. Glucose and fructose at this stage showed a 2-fold increase in the achlorophyllous and chlorophyllous leaf parts. With morphogenesis at the shoot apex, sucrose underwent a marked decrease in the concentration in the shoot tip, while glucose and fructose increased. Sucrose and the two hexoses decreased in the achlorophyllous and chlorophyllous leaf parts. In stage 11, there was an increase in the sucrose concentration in the early inflorescence and in the leaf parts. Glucose level decreased and fructose was reduced to trace concentration in the shoot tip, but there was a marked increase in glucose and fructose in the achlorophyllous and chlorophyllous leaf parts. Maltose was a quantitatively minor component of the shoot tip in all stages (0.4 to 2.9 mgg- I ); it was reduced to trace concentration during evocation. In the penultimate stage of vegetative growth (stage 7), maltose could not be detected in the achlorophyllous leaf parts; in all the other stages, maltose was present (0.7 to 3.5mgg- 1 in the achlorophyllous and 0.5 to 2.6mgg- 1 in the chlorophyllous leaf part). When the shoot apex was undergoing evocation reactions, maltose decreased to trace concentration also in the achlorophyllous leaf part, but it was present in 1 mg g-I concentration in the chlorophyllous leaf part. Raffinose occurred in the tissues of the pineapple plant in many stages, in concentration comparable with that of maltose, (0.4 to 1.5mgg- 1 in the shoot tip, 0.7 to 2.1 mgg- I in the achlorophyllous leaf part and 0.4 to 1 mgg- I in the chlorophyllous leaf part). In stage 2, however, raffinose was absent in all tissues. During evocation, raffinose was in trace concentration in the shoot tip and achlorophyllous leaf part, but it was present in appreciable concentration (0.8 mg g-I) in the chlorophyllous leaf part. Starch in the shoot tip was at all stages more abundant than total ethanol-soluble sugars, both expressed as hexose equivalent. The ratio starch: ethanol-soluble carbohydrate was in intermediate value in the hapa stage and was unaltered following planting. This ratio was maintained at a fairly high level throughout the rest of the vegetative state. At the time of evocation, the ratio abruptly decreased in the shoot tip. The ratio remained low also during morphogenesis, but increased in the early stage of inflorescence. In the achlorophyllous leaf part, starch exceeded ethanol-soluble carbohydrate in the vegetative state, except following planting of hapa and in the last stage. The ratio starch: total ethanol-soluble carbohydrate was always lower in the achlorophyllous leaf part than in the shoot tip. Marked reduction in the ratio was evident following planting and during evocation. The chlorophyllous leaf part generally resembled the achlorophyllous leaf part in the pattern of distribution of total ethanol-soluble sugars, total metabolisable carbohydrate and the ratio starch: total ethanol-soluble sugars.
z. Pjlanzenphysiol. Ed. 110. S. 429-438. 1983.
436
K. N. MADHUSUDANAN and S. NANDAKUMAR
Discussion The high concentration in which starch and soluble sugars occurred in the tissues of the hapa was, probably, to be traced to the dual means of carbohydrate nutrition, supply from the mother plant and photosynthesis by propagule. The constancy in the ratio starch: ethanol-soluble carbohydrate in stage 2 in the shoot tip and its decrease in the leaf parts suggested that the partition between starch and sugars had to be maintained constant to meet the physiological needs of the shoot tip, in these stages, but that in the leaf the emphasis was on the readily metabolizable and transportable form rather than the storage form. The marked decrease in starch and sugar content in the leaf parts and particularly in the shoot tip observed in stage 2 reflected the stress the propagule was subjected to when forced to grow as an autonomous plant. It also pointed to the biochemical basis for the adverse effect of undue storage of propagules in plant crop growth, reported by Norman (1980), since excessive loss of reserves and metabolites may not be reversed on subsequent planting. The precipitous decrease in starch concentration in the shoot tip in stage 2 was analogous to the events occurring in cotyledons/endosperm during germination and seedling development (Mayer and Poljakoff-Mayber, 1975). Throughout stages 3 to 7, there was no qualitative change in carbohydrate constituents, nor was an abrupt quantitative change evident at any stage. The absence of any abrupt changes was to be expected from the susceptibility of the plant to forced flowering at any stage, even though the fruit size depended on the growth stage at which the hormone was applied (Kerns et aI., 1936). The most significant changes in carbohydrate levels and distribution during ontogeny were those attendant on the transition of the vegetative to the reproductive state. In stage 8, the plant was fully mature and apparently receptive to the floral stimulus which was being elaborated in the interval between stages 7 and 8. Starch decreased insignificantly in shoot tip in stage 8 and markedly in stage 9, when evocation reactions were about to be initiated (stage 8), or had advanced (stage 9), irrevocably committing the apex to flowering. There was a spurt in sucrose level in shoot tip in stage 9, which was analogous to the increase in soluble sugars which Bodson (1977) observed in evoked Sinapis apex, but of much higher magnitude. Simultaneously, there was an increase in sucrose concentration in the achlorophyllous leaf part and more so in the chlorophyllous leaf part. Starch decreased in the achlorophyllous leaf part in stage 9 (P < 0.01), but increased in the chlorophyllous leaf part (P
z. Pjlanzenphysiol. Bd. 110. S. 429-438.1983.
Carbohydrate changes during development of pineapple
437
Glucose was at its lowest level in the shoot tip and fructose occurred in traces in stage 8; both increased in stage 9. In both chlorophyllous and achlorophyllous leaf part, the two hexoses increased in concentration in stage 9, either hexose being present in markedly higher concentration than sucrose. The primary purpose of glucose and fructose accumulation in the achlorophyllous leaf part in stages 8 and 9 seemed to be for transfer to the shoot tip, to meet its metabolic needs. The pattern of these changes would lead one to speculate that target cells for the flowering principle elaborated by the green leaf were present in the achlorophyllous leaf part in addition to the shoot apex. The ontogenetic pattern of distribution of carbohydrate components in the chlorophyllous leaf part suggested that the photosynthetic activity, or, at any rate, the distribution among the primary product (sucrose) and the derived products (glucose and fructose and starch) was dependent on the physiological stage of the plant, the shoot apex in particular. The pattern of maltose distribution in the chlorophyllous leaf part was difficult to reconcile with a role that it is merely a transient intermediate in starch degradation. Maltose can be biosynthesized in green leaves (Linden et aI., 1975). Its distribution pattern in the achlorophyllous leaf part and in the shoot tip was suggestive of an active physiological role for maltose in the metabolism of pineapple shoot apex, especially during evocative reactions when the sugar was reduced to trace level. To the authors' best of knowledge, this is the first time that evidence, though circumstantial, has been found for a distinctive metabolic role for maltose in a higher plant. That raffinose also played a role during the evocation reactions at the apex appeared likely from its presence in trace concentrations only in the shoot tip and achlorophyllous leaf part in stage 9. The ratio starch: ethanol-soluble carbohydrate during the developmental stages fell into a few groups. Stage 9 witnessed an abrupt lowering in this ratio in the shoot tip, suggesting an accentuation of the partition of carbohydrate in the form of readily metabolizable sugars, in preference to storage starch, at the time of floral evocation. In stage 11, when the apex of the early inflorescence axis was reverting to the vegetative state, the ratio was more than doubled, elevating it to nearly the same magnitude as in stages 1 and 2. Unlike in the shoot tip, the ratio in the achlorophyllous leaf part was frequently less than one. This was indicative of carbohydrates being held preferentially in a form readily translocated out of leaf. Total metabolizable (nonstructural) carbohydrate decreased markedly in the shoot tip during evocation (stage 9), but increased in the leaf. Acknowledgements The authors are grateful to Professor P. S. Krishnan, Emeritus Professor in Biochemistry, for his interest in the investigation and to Professor B. K. Nayar, Head of the Department, for laboratory facilities. Our gratitude is due to Professor Dr. R. Kollmann, Kiel, for helpful suggestions in the preparation of this manuscript. We are indebted to Professor C. C. Black, Georgia, at whose suggestion the test for fructosans was carried out.
z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.
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K. N. MADHUSUDANAN and S. NANDAKUMAR
References BARTHOLOMEW, D. P. and S. B. KADZIMIN: Pineapple. In: ALVIM, P. T. and T. T. KOZLOWSKI (Ed.): Ecophysiology of Tropical Crops, 113-156. Academic Press, Inc., New York, San Francisco, London, 1977. BERNIER, G.: Sinapis alba L. In: EVANS, L. T. (Ed.): The Induction of Flowering, 305-327. Macmillan of Australia, Melbourne, 1969. BODSON, M.: Changes in the carbohydrate content of the leaf and the apical bud of Sinapis during transition to flowering. Planta 135, 19-23 (1977). COLLlNS,J. L.: The Pineapple. pp. 295. Leonard Hill, London, 1968. EVANS, L. T.: Flower induction and the florigen concept. Annu. Rev. Plant Physiol. 22, 365-394 (1971). - Inhibition of flowering in Lolium temulentum by the photosynthetic inhibitor 3 (3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in relation to assimilate supply to the shoot apex. In: JACQUES, R. (Ed.): Etude de Biologie Vegetale. Hommage au Professuer P. Chouard, Paris, 1976. GIFFORD, E. M.: Initiation and early development of the inflorescence in pineapple (Ananas comosus «Smooth Cayenne») treated with acetylene. Amer. J. Bot. 48, 657-666 (1969). GOWING, D. P.: Experiments on the photoperiodic response in pineapple. Amer. J. Bot. 48, 16-21 (1961). KERNS, K. R., J. L. COLLINS, and H. KIM: Developmental studies of the pineapple Ananas com· osus (L.) Merr. 1. Origin and growth of leaves and inflorescence. New Phytol. 35, 305-317 (1936). KINET, J. M.: Gross chemical changes occurring in the leaf of Sinapis alba during photoperiodic induction of flowering. New Phytol. 74,25-32 (1975). LINDEN, J. c., N. SCHILLING, H. BRACKENHOFER, and O. KANDLER: Asymmetric labelling of maltose during photosynthesis in 14C02. Z. Pflanzenphysiol. 76, 176-181 (1975). MAoHUSUDANAN, K. N., E. NABEESA, V. UMADEVI, and S. NANDAKUMAR: Crop growth pattern and the propagule differentiation of 20 varieties in pineapple. Scientia Horticulturae 18, 215-224 (1983). MAYER, A. M. and A. POLJAKOFF-MAYER: The Germination of Seeds. pp. 192. Pergamon Press, Oxford, New York, Sec. Edn. 1975; reprinted, 1978. MONTGOMERY, R.: Determination of glycogen. Arch. Biochem. Biophys. 67, 378-386 (1957). NORMAN, J. c.: Effects of storage and type of planting material on pineapple, Ananas comosus cultivar sugarloaf. Gartenbauwissenschaft 45, 255-259 (1980). SCHWABE, W. W.: Physiology of vegetative reproduction and flowering. In: STEWARD, F. C. (Ed.): Plant Physiology, 6A, 233-411. Academic Press, New York and London, 1971. VINCE-PRUE, D.: Photoperiodism in Plants. pp. 444. McGraw-Hill, London, 1975.
Z. Pjlanzenphysiol. Bd. 110. S. 429-438. 1983.