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Changes in ribonucleic acids in citrus fruit with growth and development

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Title:
Changes in ribonucleic acids in citrus fruit with growth and development
Creator:
Barmore, Charles Rice, 1942- ( Dissertant )
Biggs, Robert H. ( Thesis advisor )
Krezdorn, Alfred H. ( Reviewer )
Humphreys, Thomas E. ( Reviewer )
Smith, Richard C. ( Reviewer )
Wiltbank, William J. ( Reviewer )
Reynolds, John E. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1972
Language:
English
Physical Description:
130 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Cell growth ( jstor )
Citrus fruits ( jstor )
Electrophoresis ( jstor )
Fractions ( jstor )
Fruits ( jstor )
Gels ( jstor )
Nucleic acids ( jstor )
Nucleotides ( jstor )
Orange fruits ( jstor )
RNA ( jstor )
Citrus -- Growth ( lcsh )
Dissertations, Academic -- Fruit Crops -- UF
Fruit Crops thesis Ph. D
RNA ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
Quantitative and qualitative changes in RNA and acid-soluble nucleotides in the fruit pulp of 'Orlando' tangelo, and 'Hamlin', 'Pineapple' and 'Parson Brown' oranges during stages M and III of growth and development were studied. Analyses of these constituents were made with an i on-exchange column chromatography, UV spectral analysis and gel-electrophoresis. It was concluded from this study that quantitative changes in RNA are gradual over the stages of fruit development examined. This conclusion was based on the following observations from total RNA and gel-electrophoretic determinations: Changes in concentration of total RNA were gradual from the mid-point of stage II until well into stage 111, with only a slight increase just prior to completion of stage II of fruit growth. Gel-electrophoretic separation of RNA yielded patterns that indicated that RNA changes were gradual decreasing until the transition from stage II to stage III and then remaining fairly constant for at least 2 months, and possibly longer. In 'Calamondin' fruit, maximum incorporation of 32p into RNA occurred also at the transition from stage II to stage Ml, and appreciable 32p activity was found in the mRNA region of the electrophoretogram . It was suggested that the increased RNA in the early part of stage II I of fruit development may be essential for the initiation of changes associated with ripening of citrus fruit, e.g., production of enzymes required for changes normally associated with ripening. Analysis of the acid-soluble nucleotide fraction of the fruit indicated that they are present in appreciable quantities in fruits and most surely play a vital role in fruit development. The nucleotides NAD, CMP, AMP, UMP, ADP, CTP, GDP, UDP, and ATP were tentatively identified based on the elution position from an an ion-exchange column and spectral analysis. ATP was further assayed by the luciferin-luciferase method. The possible significance of nucleotides in carbohydrate metabolism and in the edible fruit was discussed, particularly as nucleotides may modify flavor.
Thesis:
Thesis (Ph. D.)--University of Florida, 1972.
Bibliography:
Includes bibliographical references (leaves 122-129).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Charles Rice Barmore.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030282296 ( alephbibnum )
37680231 ( oclc )
ACJ8510 ( notis )

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Changes In Ribonucleic Acids in Citrus Fruit With Growth and Development By CHARLES RICE BARMORE A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQU I REHEi-lTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1972

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UNIVERSITY OF FLORIDA iiiiii iiiiiii 3 1262 08552 4774

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ACKNOWLEDGMENTS The author wishes to express his sincere gratitude to Dr. R, H. Biggs, chairman of the supervisory committee, for his valuable assistance and encouragement during the research and preparation of this manuscr i pt. Appreciation is also extended to Dr. A. H. Krezdorn (chairman), Department of Fruit Crops; Dr. T. E. Humphreys, Professor, Department of Botany; Dr. R. C. Smith, Assistant Professor, Department of Botany,' and Dr. W. J. Wiltbank, Assistant Professor, Department of Fruit Crops, for their constructive criticism and assistance in the preparation of thi s manuscr i pt. The author's sincere gratitude is extended to Mrs. Lillian S. Ingenlath for the expert typing and her patience during the preparation of the manuscri pt. The author also wishes to express his deepest appreciation to Mr. & Mrs. John M. Barmore for their financial assistance and encouragement.

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TABLE OF CONTEfJTS Page ACKMOWLEDGMEflTS i i LIST OF TABLES v LIST OF FIGURES vi KEY TO SYMBOLS OF ABBREVIATIONS x ABSTRACT xi INTRODUCTION 1 LITERATURE REVIEV/ 3 General Considerations 3 Morphological and Anatomical Changes During Fruit Grovjth 3 Physiological and Biochemical Changes h Nitrogenous compounds ^ Respiration 5 V/ater Uptake 7 Role of Nucleic Acids in Growth and Deve] oprr.ent . . 8 MATERIALS AND METHODS 1^ Plant Materials I^f Total Soluble Solids and Titratablc Acidity Measurement 1^ Preparation of Tissue Extracts 1^ Determination of Total RNA Concentration ... 1^ Determination of Protein Concentration and the Percent AISR 16 Isolation of Cytoplasmic Ribosomal and Soluble RiJA 17 Preparation of RtIA 17 Preparation of gels IS Procedure for electrophoresis 18 Scanning of ultraviolet absorption .... 19 Incorporation of 32p jpto RNA 19 Radioactivity determination 19 Determination of Acid-Soluble Nucleotides ... 19 Determination of ATP Concentration 21

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Page RESULTS 22 Fruit Growth 22 Percent Soluble Solids and Titratabie Acidity in Citrus Fruit Pulp at Various Stages of Growth and Development 22 RNA Concentration in Citrus Fruit Pulp at Various Stages of Growth and Development 29 Protein Concentration in Citrus Fruit Pulp at Various Stages of Growth and Development 36 Fractionat ional Patterns of Cytoplasmic RNA from Citrus Fruit Pulp at Various Stages of Growth and Development 36 Labeling of 'Calamondin' Fruit with 32p at Various Stages of Growth and Development 57 Separation of Acid-Soluble Nucleotides in Citrus Fruit Pulp at Various Stages of Growth and Development 59 Concentration of ATP in Citrus Pulp at Various Stages of Fruit Growth and Development 103 DISCUSSION no SUMmRY AND CONCLUSION 120 LITERATURE CITED 122 BIOGRAPHICAL SKETCH 130

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LIST OF TABLES Table Page 1. Experimental material I5 2. Soluble solids in citrus fruit pulp at various stages of grov-/th and development 27 3. Titratable acidity in citrus fruit pulp at various stages of grov/th and development .... 28 k. Concentration of RNA and protein in the alcoiiol insoluble residue (AISR) fraction of the citrus fruit pulp at various stages of growth and development 35 5. Protein concentration in the citrus fruit pulp at various stages of growth and development 37 6. Ratios of the cytoplasmic RtIA fractions from citrus fruit pulp at various stages of grov/th and development 58 7. Spectroscopic analysis of fractions of a knov/n mixture of 5' mononucleotides separated on a Dowex 1 X 8 (formate form) ion-exchange in a formate systeii 10'+ 8. Spectroscopic analysis and tentative identification of the acid-soluble nucleotide fractions extracted from citrus fruit pulp separated on a Dovyex 1 X 8 (formate form) ionexchange in a formate system 105 9. Estimated quantity of the acid-soluble nucleotide fractions in mg per ^0 g of citrus fruit pulp at various stages of grov;th and development IO6 10. Concentra lion cf ATP in citrus fruit pulp at various stages of growtfi and development 109

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LIST OF FIGURES Figure Page 1. Grov/th curve of 'Orlando' tangelo fruit 2k 2. Growth curve of 'Hamlin' orange fruit 2k 3. Growth curve of 'Pineapple' orange fruit 26 k, Grov/th curve of 'Parson Brov/n' orange fruit .... 26 5. Total RNA concentration in the fruit pulp of 'Orlando' tangelo during growth and development 31 6. Total RNA concentration in the fruit pulp of 'Hamlin' orange during growth and development 3' 7. Total RNA concentration in the fruit pulp of 'Pineapple' orange during growth and development 33 8. Total RNA concentration in the fruit pulp of 'Parson Brown' orange during growth and development 33 9. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Orlando' tangelo fruits harvested 7-23-70 39 10. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Orlando' tangelo fruits harvested 9-26-70 kO 11. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Orlando' tangelo fruits harvested 11-25-70 k] 12. Electrophoi-etogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Orlando' tangelo fruits harvested 12-12-70 kl 13. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Hamlin' orange fruits harvested 7-23-70 k3 vi

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Figure Page; 1^. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RMA from 'Hamlin' orange fruits harvested 9-11-70 kh 15. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RMA from 'Hamlin' orange fruits harvested 11-25-70 ^5 16. Electrophoretogram on 2.6%, acrylamide gel of cytoplasmic RNA from 'Hamlin' orange fruits harvested 12-22-70 kS 17. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Pineapple' orange fruits harvested 7-23-70 ........ kj 18. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RMA from 'Pineapple' orange fruits harvested 9-26-70 ^8 19. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RMA from 'Pineapple' orange fruits harvested 11-25-70 kS 20. Electrophoretogram on 2.S7o acrylamide ge] of cytoplasmic RNA from 'Pineapple' orange fruits harvested 12-22-70 50 21. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Pineapple' orange fruits harvested 1-17-71 51 22. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Parson Brov;n' orange fruits harvested 7-23-70 52 23. Electrophoretogram on 2.6% acrylamide ge] of cytoplasmic RNA from 'Parson Brovm' orange fruits harvested 9-26-70 53 2k, Electrophoretogram on 2.6%. acrylamide gel of cytoplasmic RNA from 'Parson Brown' orange fruits harvested 11-25-70 5>k 25. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RMA from 'Parson Brovm' orange fruits harvested 12-22-70 55 26. Electrophoretogra-. on 2.6% acrylamide gel of cytoplasmic RNA from 'Parson Brown' orange fruits harvested I-I7-7I 56

PAGE 9

Figure Page 27. Electrophoretogram on 2.6% acrylamide ge'i of cytoplasmic RNA from immature ' Cal.rno.idi n' orange fruit 1.5 cm in diameter 60 28. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from mature, green 'Calamondin' orange fruits 3.0 cm in diameter 61 29. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from very mature 'Calamondin' orange fruits 62 30. Anion-exchange chromatogram of the 5' mononucleotides separated on a Dowex 1 X 8 (formate form) ion-exchanger in a formate system 6^+ 31. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Orlando' tangelo fruit pulp harvested 8-I9-7O 66 32. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Orlando' tangelo fruit pulp harvested 9-26-70 68 33. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Orlando' tangelo fruit pulp harvested 11-25-70 70 3^. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Orlando' tangelo fruit pulp harvested 12-12-70 72 35. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Hamlin' orange fruit pulp harvested 7-23-70 Jk 36. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Hamlin' orange fruit pulp harvested 8-I9-7O 76 37. Anion-exchange chromatogram of the acidsoluble nucleotides of 'Hamlin' orange fruit pulp harvested 9-II-70 78 38. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Hamlin' orange fruit pulp harvested 11-?5-70 80

PAGE 10

Figure Page 39. Anion-exchange chromatogram of the. acid-soUible nucleotides of 'Hamlin' orange fruit pu^ri harvested 12-22-70 82^0. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Pineapple' orange fruit pulp harvested 8-19-70 84 h] . Anion-exchange chromatogram of the acid-soluble nucleotides of 'Pineapple' orange fruit pulp harvested 9-26-70 86 k2. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Pineapple' orange fruit pulp harvested 11-25-70 88 43. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Pineapple' orange fruit pulp harvested 12-22-70 90 kh. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Pineapple' orange fruit pulp harvested 1-17-71 92 ^5. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Parson Drown' orange fruit pulp harvested 8-19-70 9^ k6. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Parson Brov;n' orange fruit pulp harvested 9-26-70 96 '47. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Parson Brov;n' orange fruit pulp harvested 11-25-70 98 ko. Anion-exchange chromatogram of the acid-soluble nucleotides of 'Parson Brovm' orange fruit pulp harvested 12-22-70 100 hS . Anion-exchange chromatogram of the acid-soluble nucleotides of 'Parson Bro'yn' orange fiuit pulp harvested 1-17-71 102

PAGE 11

KEY TO SYMBOLS OF ABBREVIATIONS AISR Alcohol insoluble residue AMP Adenosine monophosphate ADP Adenosine diphosphate ATP Adenosine triphosphate CMP Cytidine monophosph-ate CDP Cytidine diphosphate CTP Cytidine triposphate DNA Deoxyribonucleic acid lAA I ndole-3-acet i c acid GA Gibberel 1 ic acid GMP Guanos ine monophosphate GDP Guanosine diphosphate GTP Guanosine triphosphate mCi Mi 1 1 i curie NAD Nicotinamide-adenine dinucleotide NADP"^ Nicotinamide-adenine dinucleotide phosphate RNA Ribonucleic acid mRNA Messenger RNA sRNA Soluble RNA tRNA Transfer RNA SDS Sodium dodecyl sulfate UMP Uridine monophosphate UDP Uridine diphosphate UTP Uridine triphosphate

PAGE 12

Abstract of Dissertation Presented to the Graduate Council of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHANGES IN RIBONUCLEIC ACIDS IN CITRUS FRUIT WITH GROWTH AND DEVELOPMENT By Charles Rice Barmore March, 1972 Chairman: Dr. R. H. Biggs Major Department: Fruit Crops Quantitative and qualitative changes in RNA and acid-soluble nucleotides in the fruit pulp of 'Orlando' tangelo, and 'Hamlin', 'Pineapple' and 'Parson Brov;n' oranges during stages M and III of growth and development were studied. Analyses of these constituents were made v;i th an i on-exchange column chromatography, UV spectral analysis and ge 1 electrophoresis. It was concluded from this study that quantitative changes in RNA are gradual over the stages of fruit development examined. This conclusion was based on the following observations from total RNA and ge 1 -e lect rophoret i c determinations: Changes in concentration of total RNA v;e re gradual from the mid-point of stage II until well into stage 111, with only a slight increase just prior to completion of stage II of fruit growth. Ge 1 -e lect rophoret i c separation of RNA yielded patterns that indicated that RNA changes were gradual decreasing until the transition from stage II to stage III and then remaining fairly constant for at least 2 months, and possibly longer. In 'Calamondin' fruit, maximum incorporation of -^ P into RNA occurred also at the transition from stage II to stage Ml, and appreciable -' P activity was found in the mRNA region of the e lect rophore togram . It was suggested that the increased RNA in the early part of stage II I of

PAGE 13

fruit development may be essential for the initiation of changes associated with ripening of citrus fruit, e.g., production of enzymes required for changes normally associated with ripening. Analysis of the acid-soluble nucleotide fraction of the fruit indicated that they are present in appreciable quantities in fruits and most surely play a vital role in fruit development. The nucleotides NAD, CMP, AMP, UMP, ADP, CTP, GDP, UDP, and ATP were tentatively identified based on the elution position from an an ion-exchange column and spectral analysis. ATP was further assayed by the luciferinluciferase method. The possible significance of nucleotides in carbohydrate metabolism and in the edible fruit was discussed, particularly as nucleotides may modify flavor.

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INTRODUCTION During grov/th and development of an organism an irreversible increase in size occurs, and there is an increase in net protein synthesis. In recent years, much v,'ori< has been directed tO'.'aid understanding this process. Yet, current physiological kno'.vledge cemnot adequately explain grov/th and development. Numerous books and reviev/s have been written on the relation of nucleic acids to biological reactions. These indicate the importance attached to this group of compounds. Yet, very little is knov;n of the relatiori of nucleic acids to the grov/th and dsvelopmen c of a fruit. Nucleic acids and their derivatives are of frequent occurrence in plant tissue. They are knov;n to be essential for p.otein synthesis; storage and transfer of genetic information; and the mediation and utilization of respiratory energy for the synthesis of rcl hilar components. An analysis of the changes of nucleic acids and their derivatives during 'grov/th and development should serve as an index of cliange in various biochemical processes. The actions of various coi'ipounds affecting growth and development have shov/n either a direct or indirect effect on nucleic acid metabolism (59). The mode of action of grov/th regulators has been suggested as being mediated through i.ucleic acid metabolin-m (5-7). Thus, an uPfdcrstandi ng of the changes of nucleic acids during this period could possibly conir i biitc to th^; understanding of the regulatory mechanisms or gro./tii and dovel opmeirL and aid in the use of grov/th regulating compounds, part i rui nr ly in ai.ering fruit physiology,

PAGE 15

This study is designed to investigate the quantitative and qualitative changes of RfJA and acid-soluble nucleotides in the grov/th and development of several citrus varieties, including both seedy and nonseedy cultivars.

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LITERATURE REVIEW General Considerations The citrus fruit is a modified berry called a hesperidum. For descriptive purposes, the fruit has been divided into 2 regions, the pericarp and the septa. The pericarp is that portion exterior to the carpels and is divided into 3 regions; the exocarp or flavedo, the mesocarp or albedo, and the endocarp, the inner portion of the pericarp and portion of the locular membrane. The edible portion of the fruit is the fused carpels containing the juice vesicles. The juice vesicles are multicellular structures originating from the locular wall, which are part of the endocarp, with a layer of cells on the surface that are small and epi de rmi s -1 i ke with a cuticle covering. The internal cells are highly evacuolated (89). Morphological and Anatomical Changes Pur i ng Fru i t Grov/th Bain (6) reported that the grovyth of the 'Valencia' orange fruit is distinguished by 3 stages, each stage being characterized by distinct morphological, anatomical and physiological changes. Stage I is the cell division stage since the number of cells in all of the tissues of the developing fruit increase to form the tissue of the mature fruit. increase in fruit size during this stage is due primarily to growth of the pericarp. During this stage, the juice sac primordium grows by cell division and forms a club-shaped head supported by a stalk. 3

PAGE 17

k Stage II is the period of rapid increase in size, primarily by cell enlargement. This stage corresponds to the maximum growth period. There is a marl<.ed expansion of the tissues accompanied by cell enlargement and differentiation during this stage. The pulp tissue increases considerably, and the juice sacs continue to divide and enlarge accompanied by an increase in juice content. Cell division is completed in all tissues except the peel at the end of this stage. Stage III is regarded as the period of maturation. This stage is readily distinguished from stage II by its decreased rate of morphological, anatomical, and physiological changes. Physiological and Biochemical Changes Nitrogenous compounds In the mature citrus fruit, nitrogenous compounds compose 5 to 10% of the total solids. The primary nitrogenous compounds found in citrus fruits are amino acids, amines, peptides, and proteins (86). Investigations into the changes of both catalytic and structural proteins during citrus fruit growth are limited. Bain (6) found that protein nitrogen Increased greatly during the cell division stage in the fruit of the 'Valencia' orange, owing to the synthesis of new cytoplasm, but the total nitrogen content remained unchanged. Throughout stage II, protein contained the bulk of the nitrogen. During maturation. Increase in protein nitrogen occurred at a decreased rate. The total protein concentration of the juice insolubles is reported to be approximately 3^% at maturity (7). Among the proteins In citrus fruits, major interest has been on enzymes, especially those that hydrolyze pectins. Pectlnase was re-

PAGE 18

5 ported to be associated with the insoluble components of the fruit and not present in the juice (51, 71). The occurrence of other enzynies such as acety lesterase (52), phosphatase (if), decarboxylase (5), peroxidase (30) and enzymes associated with mitochondria (23, Sk, 105), have also been reported. In contrast to most enzyme systems, phosphatase activity vjas also found in the filtered juice. A considerable number of amino acids have been identified as occurring in citrus fruit, and are found in all components of the fruit. During the early stages of fruit grov/th, ammonia, asparagine, aspartic acid, and serine were predominant. Ammonia declined to a negligible amount by maturity. Clements and Leland (28) found that in mature 'Valencia' fruits proline, arginine, and aminobutyric acid' were the predominant amino acids. The amino acid nitrogen accounted for 61% of the total nitrogen in the young fruit and 83% at maturity. Res pi ration Respiration data on various types of fruits generally fall into one of tv/o basic categories. Biale (12) has designated these as either climacteric fruits or non-climacteric fruits. In climacteric fruits the respiration declines throughout growth to a ]ovi level called the precl i iriocter i c minimniii, rises sharply to a climacteric maximum, and then drops markedly during the post-climacteric period. The sharp respiration increase is considered the point in the life of the fruit v/hen grov/th and maturation are complete and the stage is set for senescence and deterioration ($6). A concomitant increase in ethylene production also occurs during the climacteric (21), Associated v/ith the climacteric are a number of physical and chemical chariges vjh'ch are characteristic of tfie ripening process

PAGE 19

such as color transformation, change in pectins from insoluble to soluble forms, and transformation of starches to sugars (13, 31, ^6). These changes occur in fruits left on the tree and in harvested fruits, but the processes are accelerated with detachment if other conditions, particularly temperature, are similar (14, 41, 96). The pattern of respiration of citrus fruit is decidedly different from climacteric fruit. Citrus fruits do not exhibit a pronounced rise in respiration and are classified as non-climacteric fruits. Bain (6) followed the metabolic activity in 'Valencia' oranges from early fruit set through the stages of cell division, enlargement and maturation. The respiration rate per fruit increased during the first 2 stages of growth but declined throughout the maturation stage. Ripening occurred during the third stage without a climacteric. There is no conversion of insoluble carbohydrates, such as starches into dior monosaccari des , and the changes of insoluble protopectins to the soluble forms are not clear cut (l8). In general, the stage of maturity, or ripeness, of citrus fruits is determined on the ratio of total soluble solids to acids. The concentration of the soluble solids and sugars increases and the acids decrease in the pulp during growth and maturity of the fruit (42, 54, 94). This increase is apparently at the expense of the organic acids synthesis and changes in cell wall constituents (96). The reducing sugars comprise approximately 50^ of the total sugar with the ratio remaining fairly constant during the later stages of development. The concentration of reducing sugars is more variable than that of the nonreducing, probably due to the fact that this carbohydrate fraction is readily metabolized (94).

PAGE 20

7 Maximum amounts of free acids in 'Valencia' orange fruit v-;ere found to develop early in the season and to cinange very little up to maturity (83). Varma and Ramakrishnan (101) found that in citrus fruits having a diameter of 1,5 cm or greater citric acid was the predominant acid with small amounts of malic acid present. During stage I of fruit growth, succinic acid was the predominant one. Total acidity reached its peak in concentration in early fall and then v^as respired and/or diluted by an increase in vacuolar water as the juice vesicles increased in size and became highly vacuolated. During ripening, a further decrease in total acidity was observed (^2). The mechanism(s) responsible for acid accumulation in citrus fruits have been examined by Buslig (23). Generally, it is believed that organic acid accumulation is caused by overproduction of a specific acid vyh i ch cannot be efficiently metabolized. Buslig found that citrate is metabolized by sour varieties less efficiently than by the not-quite-so-acid fruits. This was due in part to an altered control of the enzyme citrate synthetase in the more acid fruits. The energetics of acid accumulation may be accounted for by the high levels of ATP and the high ATP/ADP ratios in sour fruits during the greatest increase in citric acid content. ATP is believed to be required for the transport of acids from the mitochondria to the vacuole for subsequent accumulation. Water Uptake Fruit enlargement is accompanied by a large uptake of water. Up to 90,' of the fresh weight can be attributed to water (75). Bain (6) reported that the enormous increase in fresh v/eight during stage 11 was primarily due to an accumulation of water in the pulp

PAGE 21

8 segments. The water content can fluctuate significantly during growth and development, especially during a period of drought. Barthalomew and Reed (8), working with lemons, found that leaves may withdraw considerable amounts of water from the fruit when water cannot be supplied by the roots. Role of Nucleic Acids in Growth and Development Nucleic acids and their derivatives are constituents of every living cell, and probably constitute one of the most important groups of compounds affecting growth and development. These compounds direct the synthesis of protein and in many instances control various metabolic reactions. The suggestion has been made that the action of growth hormones may be mediated through an affect on nucleic acid metabolism (57). As outlined in several biochemical texts (16, 29, 72), the following relationships seem to be well established. Two types of nucleic acids are known to occur in the living cell. One, DNA, i s a chief constituent of the cell nucleus, and is concerned with the storage and transfer of genetic information. The other, RNA, is contained primarily in the cytoplasm outside the nucleus, and is responsible for directing the synthesis of both catalytic (enzymes) and structural proteins. Both RNA and DNA are polymers of high molecular weight. The hydrolysis of these nucleic acids yields phosphorylated nucleotides, which consist of a ribose (RNA) or a deoxyribose (DNA) sugar, a phosphate moiety, and a nitrogenous base of either a purine or pyrimidine. The mononucleotides of RNA occur freely in the cytoplasm or combined with other constituents to form coenzymes essential for various bio-

PAGE 22

9 chemical reactions. The physiological significance of these nucleotides and coenzynes is undoubtedly great since they are essential parts of many transfer reactions. The synthesis and degradation of cellular constituents occur simultaneously in living cells. In some cases, the energy released from degradation reactions may be utilized in the synthesis of other cellular constituents, thereby maintaining an 'energy cycle'. Compounds v;hich repeatedly function in linking endogenic reactioris to exogenic reactions are the phosphorylated nucleosides and the pyridine nucleotides. Several biological processes in v/hich these compounds are of importance are: photosynthesis, respiration, and metabolism of proteins, carbohydrates, and lipids. The pyridine nucleotides, NAD and NADP and their reduced forms, are ubiquitous in the living cell. These compounds are coenzymes for the dehydrogenase enzymes v;hlch catalyze oxidation-reduction reactions. In the organic acid cycle, four of the five enzymes involved in the oxidation of pyruvic acid utilize NAD as an oxidizing agent. The pentose phosphate pathv/ay is an important source of the reduced form of NADP (NADPH) , v;hich is required for the synthesis of fatty acids and some amino acids. in both the glycolytic cycle and the photosynthat i c carbon reduction cycle, the reduced and oxidized forms of HAD and NADP ere ut i 1 i zed. The phosphorylation reactions are responsible for the production of energy rich compounds, AlP, GTP, etc. Tiiis process also utilizes both phosphorylated nucleotides and pyridine nucleotides. The pyridine nucleotides are important i ntcrmedi otes in the election transport system of oxidative phosphorylation. These compoinds are essential for the metabolism of carbohydrates, proteins, lipids, and nucleic acids.

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10 Synchronization of the numerous biological reactions requires control mechani sn;5. In many reactions, various phosphory lated nucleotides and pyridine nucleotides could serve this specific function. In glycolysis the rate of the reactions catalyzed by qlyceiuldehyd2-3phosphate dehydrogenase and pyruvic kinase is detenni n&c! by the relative amount of ADP present. The activity of tvio enzymes, glycogen phosphorylase and phosphof ructoki nase, is regulated by AMP. AMP also serves as a regulator of the enzyn^e isocltrate dehyc'r ogenase in the citric acid cycle. ATP is a negative effector of the onzyme phosphof ructoki nase (17, 29). Probably, the most well defined relation betv/een plant growth and development and nucleic acid metabolism is in the syniiiesis of protein. Both cell division and cell elongation are accompanie:' by a synthesis of both structural and catalytic protein (53, 56, 85). The current concept is that protein synthesis requires 3 distinct kinds of polyn;eric RtJA; rRNA, sRMA and mRMA. Phosphoryl ated nucleotides are also essentia] in the energy transfer reactions. The different species of RMA required have been isolated from plant tissue and identified with the use of gel electrophoresis ( hj , hS , 68, 69, 103). E lectrophoret i c analyse shov; rRMA and sRMA. -le third RNA component essential for protein synthesis, mRMA, is '. readily distinguishable on el ectrophoretograms v/ith UV analyses boc^'use of the amount present in the cell. Hov;ever, it Is distinguishable v/ith radioactive labeling. Tv.'o types of rRMA are found in the green plant tissue, cytoplasmic rRtlA and chloroplas ti c rRMA. The latter is found primarily in photosynth.'^t ic tissue. The RMA of each of these riboso;nal units appears as a distinct fraction having a specific molocLilar v-yeight. Tfie rRMA of the cytoplasm is

PAGE 24

comprised of a 25S and 18S RNA subunit and the chloropl esti c rRNA comprised of a 23S and 16S RDA subunit. The 2 RNA iubuoits combine with protein to form the ribosome (3). , The synthesis of protein involves several steps. The initial step of protein synthesis involves mRIIA. The genetic information residing in the DNA molecule is transferred to the mRNA. The mRfIA attaches to the ribosome and directs the synthesis of protein. The transfer of the amino acids to the protein synthesizing site is accomplished via attachment of a specific tRMA. The attachfii:;nt and subsequent release of the amino acids require both enzyn 3s and high energy nucleotides (26) . In recent years, numerous papers and reviews have been compiled on the relation of nucleic acid mietabollsm to the action of plant horiTiones (3'!, 57, 58, 59, 7h , 88). Furtherm,ore, many vjorkers have interpreted these results as indicating a dependency o:i nucleic acid metabolism for the manifestation of biological response to plant hormones. Horm.ones appear to affect the kind and amount of nucleic acids being synthesized by the plant (57). Silbcrger and Skoog (93) in 1953, first demonstrated that auxin affects nucleic acid metabolism. Since then, the importance of RN'A metabolism in auxin action has been confirmed by a nuniber of v;orkers ('lO, 60, 61, 108). Key and Shannon (61) observed th^it jAA at concentrations that promoted cell elongation in excised soylican h.ypocotyl tissue enhanced the incorporation of C -nucleotides Into PvIiA. lAAinduced RNA synthesis has been reported to be required for cellulaso synthesis and lateral cell expansion (32). There are several rc:ports on the character izat ion of the nev/ly synthesized RIIA induced by auxin. These

PAGE 25

12 studies indicate that auxin brings about the synthesis of all the major classes of RNA including DNA-RNA complex (59)Evidence has been accumulated by several v/orkers vvh i ch indicates that plant tissues are capable of incorporating cytokinins into RNA (35, 36). The occurrence of cytokinins in certain species of tRNA suggests that cytokinin action could possibly be connected to t rans 1 at i ona 1 processes of protein synthesis. However, there is no evidence that cytokinin activity in growth regulation is associated with its presence in tRNA (57)Investigations on the sites of action of the gibberellins have shown that gibberellins affect almost all plant tissues and organs (8l). Available information does Indicate a possible interrelationship with nucleic acid metabolism. Like other grov;th regulators gibberellln has been found to enhance the synthesis of RNA (2^) and the incorporation of labeled precursors into RNA (25, 33, 3^)These effects are primarily associated with the retardation of senescence (10, 33, 3^, 50) and the synthesis of a-amylase (80, 102). Several studies have shown that the level of RNA could possibly be used as an indicator of the metabolic condition of cells at various stages of development and senescence (50, 73, 95, 99, 103). In general a transition from an active to a more quiescent metabolic state is reflected in a decline in RNA synthesis. The most drastic changes in RNA are seen in rapidly senescing tissue. Changes associated with senescence can be controlled to some extent with the use of various growth regulators, e.g., GA and cytokinins. The action of these compounds is thought to be via maintenance of the functional state of RNA (33, 3^, 50, 79). Ku and Roman i (63) reported that the onset of senescence in the pear fruit is associated with a rapidly decreasing

PAGE 26

13 rate of ribosomal turnover which leads to a complete cessation of rRNA and ribosor.ial synthesis as fruits reach the cl imacuei i ,. Richmond and Biale {8k) have suggested that the critical intercellular transitions occurring during the latter phase of the climacteric oiay stem from a reduction of RfiA synthesis. The objective of this study v/as to investigate tlie quantitative and qualitative chances of RflA and acid-soluble nucleotides during stages II and I I I of citrus fruit growth and develoj^rrfint. Changes in these constituents could serve as an Index to the various biochemical and physiological processes associated with citrus fruit growth, maturation and senescence.

PAGE 27

MATERIALS AND METHODS Plant Materials Citrus fruit varieties, listed in Table 1, were harvested from a commercial planting at Citra, Florida, and from a planting at the University of Florida, Gainesville. Fruit were collected periodically to encompass varying physiological ages from the early portion of stage II to well into stage Mi (6). Samples of each variety vjere collected to minimize locational differences by sampling in pre-selected quadrants of each tree each time. Samples were quick frozen with dry ice soon after harvesting. Fruit growth was measured as an increase in vol ume. Total Soluble Solids and Titratable Acidity Measurement Total soluble solids were determined using a Bausch and Lomb ref ractometer , model ABBE 3L, and acidity was determined by titration. Preparation of Tissue Extracts Frozen fruit were peeled, sectioned and ground with a mortar and pestle in dry ice. The powdery pulp was kept frozen until needed. Determination of Total RNA Concentration Numerous procedures have been used for the extraction of RNA from plant and animal tissue, but most are based on the use of acids (100), phenol and a suitable buffer (68), and neutral salts (76), or a combination of these extractants. in this study, the extraction procedure

PAGE 28

15 Descr i pi;ion Ci trus reti cul uta x _C. paradi si £' si nensi s (L.) Osbeck £• si nensi s (L.) Osbeck _C. si nensi s (L.) Osbeck C. madurensi s Lou. Table 1. Experimental material Type 1. 'Orlando' tangelo 2. 'Hani i n' orange 3. 'Pineapple' orange k. 'Parson Brown' orange 5. ' Cal arr.ondi n ' orange

PAGE 29

16 of Holdgate and Goodwin (^5) was used for quantitation of total RNA. The technique of Holdgate and Goodwin permitted reliable estimation of RNA in citrus fruit pulp without the use of elaborate and time consuming procedures. Frozen pulp tissue was combined with cold 85% methanol and macerated in a Sorvall stainless steel omni-mixer (5 min at 8,000 rpm) , and extracted as follows: Frozen Pulp Macerated in cold 85% methanol Centrifuged 22,000 Xg 15 min I ' Resi due 10% Trichloroacetic acid 95% Ethanol (2X) Ethanol :Chloroform (3:1) (2X) Ethanol:Ether (1:1) Acetone 1 Supernatant I Di scard Residue 0.2N Sodium acetate (pH 6.1) Steep 12 hr (25 C) 1 Supernatant I Di scard Fi Urate The concentration of RNA was measured by scanning (220 nm to 320 nm) the RNA extract using a Beckman DB-G spectrophotometer. Yeast RNA (P-L Biochemical Laboratories) was used as a standard. Determination of Protein Concentration and the Percent AISR Frozen pulp was combined with cold 95% ethanol and macerated in a Sorvall stainless steel omni-mixer (5 min at 8,000 rpm) kept below k C. The subsequent extraction procedure was as follows:

PAGE 30

17 Frozen Pulp Macerated in 95/' cold ethanol Centrifuged 22,000 Xg 15 nin Res! due Washed wi th cold 85% ethanol (2X) Fi 1 tered Residue O.IN NaOH Steep 12 hr (35 C) Supernatant Di scard Supernatant I Di scard Filtrate Protein concentration in the final filtrate vyas measured by a modified Lowery method described by Potty (82). Bovine serum albumen v;as used as the standard. The percent AISR v/as also determined from this fraction. Isolation of Cytoplasmic Ribosomal and Solubl e RNA Pre paration of RNA A modified procedure described by Loening (68) was used. Frozen fruit pulp v/as ground in a phenol -buffer medium in a Sorvall stainless steel omni-mixer (5 niin at 8,000 rpm) l
PAGE 31

18 Aqueous phase Precipitate RNA witii cold 95% ethanol (3:1) Steep 1 hr 10 C Centrifuge 22,000 Xg 15 min Phenol phase Di scard Pellet Redissolve with 0.2% SDS + 0.15M sodium acetate (3tl) Repreci pi tate with cold 95% ethanol (3:1) Centrifuge 22,000 Xg 15 min Pellet Wash with cold 85% ethanol (2X) Centrifuge 22,000 Xg 15 min RNA pellet The RNA pellet was redissolved in 2.5 ml of the electrophoresis buffer containing 6% sucrose (RNAase free) and 0.2% SDS. This susfsension was used for electrophoresis. Preparation of gels Gels (7.5 X 0.6 cm) of 2.6% polyacrylami de were prepared according to Loening (68). Gel materials were obtained from Bio-Rad Chemical Corporation. Gels were stored in the electrophoresis buffer overnight before using. Procedure for electrophoresis The buffer for electrophoresis was prepared according to Loening (68). Electrophoresis was carried out at room temperature. Current (5 mAmps/gel) was applied for 1 hr to the gel to remove the polymerization catalyst and other impurities. The RNA suspension (50 ul) was layered on the gel and electrophoresis was continued. Suitable separation of the rRNA subunits and sRNA was obtained in 70 min.

PAGE 32

19 Scanning of ultraviolet absorption LA/ absorption zones on gels were determined using a Beckman DU spectrophotometer equipped with a Gilford Gel Scanner (Model 2000). Gels v^/ere scanned at 260 nm at a rate of 2 cm/mi n v;ith a slit width of 0.35 mm. •32 incorporation of "^ P into RNA Calamondin fruit, 1.5 and 3.0 cm in diameter at completion of fruit growth and before color break, and mature fruit prior to abscission, were labelled with -^ P orthophosphate applied to the stem end for 24 hr. The fruits were quick frozen with dry ice and the RNA extracted as previously described. Radioactivity determination Gels were frozen on a glass plate with dry ice. The ^^P-label 1 ed gels v;ere cut into 2 mm sections using a micrometer equipped with a guillotine cutting apparatus. The sections vyere digested in 0.5 ml of 30% hydrogen peroxide at 70 C overnight in scintillation vials. Counting was done in 12 ml Aquasol (New England Nuclear) using a refrigerated Packard Tri-Carb scintillation counter, mode 1 31^ EX. Determination of Acid-Soluble Nucleotides Frozen pulp vyas combined with cold 0.6 N perchloric acid and ground in a Sorvall stainless steel omni-mixer at k C. The macerate was steeped for 3 hr at i+ C and the following extraction procedure was fol lov/ed: Frozen Pulp 0.6 N Perchloric acid (3: 1) Filtered through bed of Polyclar AT (GAF Corporation) (2X)

PAGE 33

I Fi Urate pH adjusted to 5.5 with 15% KOH Steeped 3 hr {k C) Fi Urate Steeped in aci d-washed, activated charcoal (Darco G6) Charcoal Nucleotides eluted off with hot 50% ethanol and 1% NH^OH Acid soluble nucleotides 20 Polyclar AT Discard 1 Fi Urate I Di scard Citrus pulp contains large quantities of polyphenols, and removal of these compounds with Polyclar AT was required for maximum resolution with UV analysis. Removal of the acid-soluble nucleotides by Polyclar AT, a weak an ion-exchange resin, was insignificant as determined using radioactive standards to check the procedure. The final extract was reduced to 10 ml and placed onto a Dowex 1X8 (200-400 mesh) formate ion-exchange column (0.9 cm x 13 cm). Chromatography vjas begun with a 2-step elution system using 2 chambers to produce a curvilinear gradient. For range 1, the mixing chamber initially contained 250 ml of distilled water and the reservoir 250 ml of 4 N formic acid. For range 2, the mixing chamber contained 250 ml of 4 N formic acid and the^ reservoi r 250 ml of 1.6 N ammonium formate. The eluate from the column was continuously monitored at 260 nm using a Gilford analyzer (Model 2000) fitted to a Beckman DU spectrophotometer. Any material not adsorbed onto the column was eliminated by washing with distilled water prior to beginning the gradient. The 5' mononucleotides (P-L Biochemical Laboratories) vjere used as standards.

PAGE 34

21 Tentative identification was made by comparing the relative position of elution from the column with standards fractionated by the same procedure. Further identification was made by UV absorption spectral analysis and thin-layer chromatography where possible. Thinlayer chromatographic analysis was made using avicel plates prepared in the laboratory and developed in a solvent system consisting of isobutyric acid, cone, ammonium hydroxide, and water (57:^:39 v/v) . Qualitative determination was made by UV visualization. Determination of ATP Concentration Concentration of ATP in the fruit pulp was measured by a modified firefly 1 uci fer i n-1 uci ferase system described by Buslig and Attaway (2^). Each batch of firefly extract was standardized with known amounts of ATP.

PAGE 35

RESULTS Changes in soluble solids, titratable acidity, and fruit volume were determined as reference points for the work on nucleotides and nucleic acids. Fruit Growth The flowering period of the k varieties studied was between March 9 and March 30, 1970. The growth curves of these varieties are presented in Figs. 1, 2, 3, and k. Fruit size increased until early November for 'Orlando' tangelo, mid-November for 'Hamlin' and 'Pineapple', and late November for 'Parson Brown' oranges. The stages of fruit growth of citrus have been studied by Bain (6), This study was concerned with changes occurring during stage II, cell enlargement, and into stage III, fruit maturation. Percent Soluble Solids and Titratable Acidity in Citrus Fruit Pulp at Various Stages of Grovjth and Development Sugar accumulation increases with growth. The increase in sugars has been suggested to take place at the expense of cell wall constituents and decreased acid synthesis (96). The percent soluble solids and titratable acidity during fruit growth and development of each variety studied are presented in Tables 2 and 3, respectively. The major constituents of the soluble solids are the sugars, primarily sucrose, glucose and fructose (9^). As shown in Table 2, the percent soluble solids increased during the latter part of stage II and early 22

PAGE 36

Fig. 1. Gro.7th curve of 'Orlando' tangelo fruit, Fig. 2. Growth curve of 'flijfnlin' orange fruit,

PAGE 37

24 160

PAGE 38

Fig. 3. Growth curve of 'Pineapple' orange fruit. Fig. A. Growth curve of 'Parson Brov/ri' orange fruit

PAGE 39

26 WAY JVUE JULY AUG SEPT OCT f,'OV DEC JAfJ HARVE ST DATE WAY JUNE JULY AUG SEPT OCT NOV DEC JAfi HARVEST DATE

PAGE 40

27 CsJ , — r-j —

PAGE 41

28 Table 3. Titratable acidity in citrus fruit pulp at various stages of grovjth and development Harvest

PAGE 42

so ac 29 part of stage 1 ! 1 of fruit growth. These results are in agreement with those reported by others (9. kl) . The relatively high soluble lids level occurring on November 25, 1970, for all varieties is ttributed to an extended warm period preceding this date. The acidity in citrus fruit pulp is due principally to citric id with small amounts of malic, tartaric and succinic acid also present (15). Citric acid concentration was greatest in young developing fruits, decreasing with fruit growth (Table 3). The decrease in acidity was more rapid in the early part of stage 11, July 8, 1970. to October 10, 1970. i^A Concentration in Citrus Fruit Pulp at Various Stages of Growt h and uevelopment Since RNA is required for protein synthesis, determinations were made of the RNA fraction. The relative concentration in mg/100 g pulp in the fruit pulp of the i* varieties during growth and development are shown in Figs. 5, 6. 7, and 8. The RNA concentration in all varieties decreased with growth and development during the period studied. The greatest change in concentration occurred in the fruits of 'Orlando' tangelo and 'Parson Brown' oranges. The RNA concentration range from young fruit to mature fruits was 72 to 1^ mg for .Orlando' tangelo and 101 to 10 mg for 'Parson Brown' orange, respectively. The concentration range for 'Hamlin' orange fruit pulp was 82 mg in young fruit to 1^2 mg in mature fruit, and for 'Pineapple' orange 80 mg in young fruit to 20 mg in mature fruit. The concentration of RNA of 'Hamlin', 'Pineapple and 'Parson Brown' orange fruits fluctuated appreciably during the period studied in contrast to that observed in fruits of 'Orlando' tangelo. Trees of the former 3 varieties were subjected to significant

PAGE 43

Fig. 5. Total RNA concentration in the fruit pulp of 'Orlando' tangelo during grov;th and development. Fig. 6. Totc'il RNA concentration in the fruit pulp of 'j-lamlin' orange during growth and deveiopmsnt .

PAGE 44

31 Q o

PAGE 45

Fig. 7. Total RNA concentration in the fruit pulp of 'Pineapple orange during grov-jth and development. Fig. 8. Total RNA concentration in tlie fruit pulp of 'Parson Brown' orange during gro\vth and development.

PAGE 46

33 JULY AUG l-i A R V i^ S T D A T l^ o o

PAGE 47

3^ fluctuations in the soil moisture during this period. Such conditions generally resulted in variations in the water content of the fruit. Barthalomew and Reed (8) have shown that fruits serve as a source of water for leaves under drought conditions. This would alter the concentration of the various cellular constituents. in view of the fact that the water content varies with fruit growth, the RNA concentration based on the alcohol insoluble residue fraction (AISR) was also determined. All RNA is precipitated, along with other cellular constituents, by ethanol . These results are presented in Table k. Fruits of 'Orlando' tangelo showed a gradual decrease in the RNA concentration with fruit growth and development ranging from 23-7 mg in young fruits to 11.2 mg/g AISR in mature fruits. The RNA concentration in 'Hamlin' orange fruits was constant between the harvest dates of July 23, 1970, to October 10 , 1970, at approximately \3.k mg and decreasing to 13.2 mg prior to completion of fruit growth, at which time an increase occurred. The maximum concentration at maturity for the period studied was 2^.3 mg. The concentration of RNA at the various stages of growth was similar for both 'Pineapple' and 'Parson Brown' fruits. The RNA concentration was constant prior to an increase that occurred at the early part of stage III. The average concentration prior to the increase was 21.1 mg/g of pulp for 'Pineapple' orange and 20.3 mg/g of pulp for 'Parson Brown' orange. At maximum accumulation, the RNA concentration was greater in fruits of 'Parson Brown' reaching a level of 48.5 mg in comparison to 26.6 mg in fruits of 'Pineapple' orange. Following this increase, the RNA concentration decreased in both varieties to 22.9 mg in 'Pineapple' and 7-9 mg in 'Parson Brown' oranges on the last date sampled, January 17, 1971.

PAGE 48

. — \Q — LAOOOrACTi O tNJ r-CC' C\0 — — CN — D cn +-> ^^ O cn -d" CO vO CT\ CTi vo LA cn CO oA r-~r^ CSJ CM CN CM CA OA CA -^ CNJ CPl — CO LAOD rAvD rACNOJ-OOOvOCO LAr~~-vDf^O LAr^OO — — — CNCNCSICNCACA r d" — LA r^ CN JrACO vO LA LA — CM j--:l-cN_j-r^oA\£>ocn o cn cn r^ -f oa oa fA CN — r— . VO—
PAGE 49

36 Protein Concentration in Citrus Fruit Pulp £il Vcirio'jc Stages oi GrowLh onJ Dl:v c I yjp. -n L Protein synthesis has been shown to be essential for phases M and I I I of fruit growth and developnr,ent. The protein concentration in the pulp v;as determined since protein synthesis is regulated by RNA, The protein concentration based on the fresh weight of the fruit pulp is sho'wn in Table 5 for the k varieties. The protein concentration decreased during fruit growth reaching a constant level at completion of fruit growth in all k varieties. The concentrations in g/100 g of pulp v;ere similar for both 'Orlando' tangelo and 'Hamlin' orange fruits ranging from 0.78^ g and 0.756 g in young fruit to 0.492 g and 0.501 g in mature fruit, respectively. The concentration range in fruits of 'Pineapple' and 'Parson Brown' oranges were also similar ranging from 0.706 g and O.706 g in young fruit to O.5OI g and 0.^93 g in mature fruit, respectively. The protein concentration in the AISR fraction of the k varieties is presented in Table k. The protein concentration began increasing approximately 2 months prior to completion of fruit growth, reaching a constant level at this time. The increase in protein was not as great in fruits of 'Orlando' tangelo, ranging from 261 mg in young fruit to 379 mg/g AISR in mature fruit. In fruits of 'Hamlin', 'Pineapple', and 'Parson Brov;n' oranges the protein concentration ranged from a minimum of 180 mg, 153 nig and 1^2 m,g in young fruit to a maximum of 295 rng, 368 mg and 379 mg/g of AISR in mature fruit, respectively. Fract ionat ional Patterns of Cytoplasmic RNA from Citrus Fruit Fulo at Various Scaces of Growth and Developi-ent Recently, it has been demonstrated that certain changes in RNA may

PAGE 50

37 Table 5. Protein concentration in the citrus fruit pulp at various stages of growt^^ and development Harves t Date Protei n g/100 g Harvest

PAGE 51

38 be good indices for cellular states of synthesizing or non-synthesizing systems (70, 8k, 103). Electrophoretograms of polymeric RNA indicate that growing and mature fruits have markedly different polymer patterns, The RNA patterns of the k varieties studied are shown in Figs. 9 thru 26. It is assumed that the cytoplasmic rRNA subunits, 25S (Rf 0.23) and 18S (Rf 0.35), and sRNA (Rf 0.76) are the RNA species being separated in the electrophoretograms. In young fruit harvested during July and September, 2 minor components were present immediately following the 2 cytoplasmic subunits corresponding to the 23S (Rf 0.28) and I8S (Rf 0.41) subunits of plastid rRNA. The identity of the 2 minor components preceding the 25S subunit are unknown. DNA is reported to occur in this region (69). The electrophoretograms show that during fruit growth and development both the number of components and the quantity of each component decreased in all varieties. Similar results have been reported in other types of plant tissue {kj , 66, 68, 73). The possibility that RNA could have been degraded during extraction is ruled out since low temperatures and SDS, an enzyme inhibitor, were used throughout the extraction procedure. It is of special interest that plastid rRNA subunits appear to be present in young fruit but absent from mature fruit. The presence of these subunits indicate that plastids are present in the juice vesicles during the early stage of growth. The quantitative change is also noteworthy, because such changes indicate degradation. The electrophoretograms indicate that the quantity of each component decreased with growth reaching a constant level at completion of stage M. Good criteria of the metabolic state of RNA are the ratios of the major polymeric RNA fractions. Several studies indicate that the ratio

PAGE 52

39 o o Fig. 9. Electrophoretogram on 2.67. acrylamide gel of cytoplosniic RNA from 'Orlando' tangelo fruits harvested 7-23-70. Electrophoresis was carried out for 70 minutes at 5 mAmps/gel .

PAGE 53

ko o Rf Fig. 10. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Orlando' tangelo fruits harvested 9-26-70. Electrophoresis v.ts carried out for 70 minutes at 5 niAmps/gel .

PAGE 54

^1 o o .'oO 1.0 Fig. 11. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic FlNA from 'Orlando' tangelo fruits harvested 1 1-25-70. Electrophoresis v/as carried out for 70 niinntcs at 5 niAmps/gel .

PAGE 55

kl o to n ^' Fig. 12, Electrophoretogram on 2.6% acrylnmlde gel of cytopl asni c RNA from 'Orlando' tangelo fruits liarvested 12-12-70, Electrophoresis was carried out for 70 minutes at 5 mAmps/gel .

PAGE 56

h3 o w ki Fig. 13. Electrophoretogram on 2.6% acrylamidc ge] of cytoplasmic Rt!A from 'Hamlin' orange fruits harvested 7-23-70. Electrophoresis v/as carried out for 70 minutes at 5 mAmps/gel .

PAGE 57

kk o ID Rf Fig. ]k. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Hamlin' orange fruits harvested 9-11-70. Electrophoresis v;as carried out for 70 minutes at 5 mAinps/ge] .

PAGE 58

h5 kj L^ .5 1.0 Fig. 15. Electrophoretocjram on 2.6% acrylamide ge] of cytoplasmic KIIA from 'Hamlin' orange fruits harvested 11-25-70. Electrophoresis was carried out for 70 minutes at 5 n^mps/gc] .

PAGE 59

kS o to R^ Fig. 16, E]ectrophoretograr,-i on 2.6% acrylamide gel of cytoplasmic RNA from 'Hamlin' orange fruits harvested 12-22-70. Electrophoresis was carried out for 70 minutes at 5 mAmps/gel ,

PAGE 60

hi o Fig. 17. Electrophoretogram on 2.6% acrylan.icis gel of cytoplasmic RMA from 'Pineapple' orange fruits harvested 7-23-70. Electrophoresis vias cairiecJ out for 70 minutes at 5 mAmps/gel .

PAGE 61

k8 Rf Fig. 18. Electrophoretogram on 2.6% acrylaniide gel of cytoplasmic RNA from 'Pineapple' orange fruits harvested 9-26-70. Electropfioresis was carried out for 70 minutes at 5 niAmps/gel .

PAGE 62

k9 ki Fig, 19. Electrophoretogram on 2.6X acrylamlde ge] of cytoplasmic RNA from 'Pineapple' orange fruits harvested 11-25-70. Electrophoresis vjas carried out for 70 minutes at 5 niAmps/gel ,

PAGE 63

50 o CO Fig. 20. Electrophoretogram on 2.67= aery lanii de gel of cytoplasmic RNA from 'Pineapple' orange fruits harvested 12-22-70. Electrophoresis was carried out for 70 minutes at 5 mAmps/gal .

PAGE 64

51 o to CvJ Rf Fig. 21. E lectrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Pineapple' orange fruits harvested 1-17-71. Electrophoresis v/as carried out for 70 minutes at 5 mAmps/ge] .

PAGE 65

52 o R^ Fig. 22. Electrophoretogram on 2.6% acrylamide ge] of cytoplasmic RNA from 'Parson Brown' orange fruits harvested 7-23-70. Electrophoresis was carried out for 70 minutes at 5 mAnips/gel .

PAGE 66

53 o o Rr Fig. 23. Electrophoretogram on 2.6% acrylainide gel of cytoplasmic PvMA from 'Parson Brov;n' orange fruits harvested 9-26-70. Electrophoresis v-;as carried oi't for 70 minutes at 5 niAnips/ge 1 .

PAGE 67

5^ o Rf Fig. 2k. Electrophoretogram on 2.6% acrylamide gel of cytoplasmic RNA from 'Parson Brown' orange fruits harvested n-25-70 Electrophoresis was carried out for 70 minutes at 5 mAmps/ge] .

PAGE 68

55 o CD Rf Fig. 25. Electrophoretogram on 2.6% acrylamide gc] oF cytoplasmic RtlA from 'Parson Brown' orange fruits Piarvestcd 12-22--70, Electrophoresis was carried out for 70 minutes at 5 niAmps/ge] .

PAGE 69

56 o ki Rf Fig. 26. Electrophoretograni on 2.6% acrylaniide gel of cytoplasmic RNA from 'Parson Brown' orange fruits harvested I-I7-7I. Electrophoresis was carried out for 70 minutes at 5 niAmps/gcl .

PAGE 70

57 of the cytoplasmic rRNA subunits, 25S:l8S, is usually 1.8-2.0 in various types of tissue. Ratios deviating from this would indicate an increase in the percent turnover of RNA {k7 , 103). The ratios of the major polymeric RNA fractions are presented in Table 6. The trends of the cytoplasmic rRNA subunit ratios were similar in fruits of 'Hamlin', 'Pineapple', and 'Parson Brown'. The ratios increased with fruit growth from 1.25 to 1.66 in 'Hamlin' fruit, 1.00 to 1.78 in 'Pineapple' fruit, and 1.00 to 2.00 in 'Parson Brown' fruit. However, in 'Orlando' tangelo fruit, the ratios of the same subunit fruits did not show an increase until December 12, 1970, at which time the ratio was 1.6. The ratios of the rRNArsRNA followed the same trend as the rRNA subunits for each variety. These data suggests that the percent turnover of cytoplasmic RNA decreases with growth becoming rather constant at the completion of stage II of fruit growth. Labeling of 'Calamondin' Fruit vjith ' P at Various Stages of Grovjth and Development •3 To ascertain further the nature of the nucleic acid system, -^ P was applied to fruit at 3 different stages of growth and development. Preferential synthesis of specific RNA fractions has been shown to occur concomitantly with changes in the metabolism of various plant organs (50, 77). 'Calamondin' fruit of 3 physiological ages were labelled with 32p applied through the stem end for 2k hr. 'Calamondin' fruit were chosen as experimental material since all 3 ages could be obtained from the same plant at the same time. The RNA was fractionated by gelelectrophoresi s and the ^^P activity in the various regions of the gel determined by scintillation counting. The electrophore t i c patterns of -^ P incorporation into RNA of fruits at different physiological ages

PAGE 71

58 Table 6, Ratios of the cytoplasmic RNA fractions from citrus fruit pulp at various stages of growth and development Harvest Harvest Date 25S:18S rRNA:sRNA Date 25S:l8S rRNArsRNA 'Orlando' tangelo 'Hamlin' orange 7-23-70 1.02 1.02 7-23-70 1.25 0.6i+ 9-26-70 1.00 1.00 9-11-70 1.38 1.^1 11-25-70 1.22 1.11 11-25-70 1.62 1.57 12-12-70 1.60 1.33 12-22-70 1.66 1.67 'Pineapple' orange 'Parson Brown' orange 7-23-70

PAGE 72

59 are presented in Figs. 27, 28, and 29. The major UV absorbing RNA components vjere cytoplasmic rRNA and sRNA. It must be pointed out that counting of 2 mm gel slices did not provide a radioactive profile vjith the same degree of resolution found in the UV absorbance scans. 32 Incorporation of P occurred in young fruits and fruits having completed their increase in size but still green. The most noticeable feature of these radioactive profiles is the high rate of incorporation •JO by green fruits which had completed growth and the absence of -'"'P incorporation by mature fruit prior to abscission. In the electrophoretograms of young fruit, P activity was found in 3 regions, rRNA, mRNA, and sRNA. Maximum activity in these regions were 1,250 cpm, 1,^70 cpm, and 1,550 cpm, respectively. The activity in the mRNA region of the electrophoretogram is of special interest. This particular RNA species has been suggested as being a DNA-RNA complex {hS, kS , 50), and is an important intermediate in protein synthesis (M+) . Separation of Acid-Soluble Nucleotides in Citrus Fruit Pulp at Various Stages of Grovjth and Development Much evidence has been accumulated indicating the importance of nucleotides in plant metabolism, particularly carbohydrate metabolism. Therefore, the fruit pulp was examined for nucleotide content. With some minor changes, extraction and analyses were similar to those used for other types of tissues (18, 90). A typical ion-exchange chromatogram of the 5' mononucleotides is shown in Fig. 30. Overlapping of several of the nucleotides, AMP-CDP, CTP-GDP, and UDP-ATP, is a common occurrence vjith this type of analysis. The elution sequence of the acid-soluble nucleotides in the fruit pulp of the k varieties are presented in Figs. 31 through kS . Identification of the various fractions

PAGE 73

60 Fig. 27. Electrophoretogram on 2.6% acrylan-iide ge] of cytoplasmic RNA from immature 'Calamondin' orange fruit 1.5 cm in diameter. Fruits v;ere labeled with 32p orthophosphate (0.01 mCi /fruit) applied to tj-ie stem end for 2h hours. Electrophoresis was carried out for 70 minutes at 5 mAmps/ gel. , E250; -• . Radioactivity.

PAGE 74

61 2000 o r, •^" R-; Fig. 28. E Icctrophoretogram on 2.6?o acrylamide gel of cytoplasmic RNA from mature, green 'Calamondin' orange fruits 3-0 cm in dirimctcr. Fruits v/e re labeled with ^^P orthophosphate (0.01 mCi/fruit) applied to the stem end for 2'i hours. Electrophoresis was carried out for 70 minutes at 5 mAmps/ gc -260: Radi oact I V i ty .

PAGE 75

62 o < q b < Fig, 29. E lectrophoretogram on 2.6% acrylaMide gel of cytoplasmic RNA from very mature 'Calamondin' orange fruits. Fruits were labeled with 32p orthophosphate (0.01 mCi /fruit) applied to the stem end for 2k hours. Electrophoresis vias carried out for 70 minutes at 5 mArnps/gel. , E26O' ' Radioactivity.

PAGE 76

in

PAGE 77

6k O O < 00?;

PAGE 78

O E O — i_ +j Q) o 014XI <+— o X CD ul 0) 0) 5 " O O T) .Q — 4-> U E

PAGE 79

66 O O <

PAGE 80

—

PAGE 81

68 O >^i o < 092 3

PAGE 82

03

PAGE 83

70 O < L!9-,. J

PAGE 84

^«

PAGE 85

72 o < 0S2_y

PAGE 86

.

PAGE 87

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103 was based on their relative position of elution, 280:260 and 250:260 nm ratios and Emax (Tables 7 and 8), and TLC analysis where possible. The presence of residues from the elution solvents interfered with TLC analysis of many of the fractions. The position of elution matched very closely the position of several standard nucleotides. However, comparison of their absorbance ratios and Emax to those of the standards indicate they are not the same compounds. Also, the Rfs of several of the major fractions from TLC analysis did not correspond to any of the Rfs for the standards. However, the presence of other UV absorbing compounds can cause a shift in the UV absorption spectrum. Tentative identification of several of the fractions based on elution position from the column is presented in Table 8. (Quantitative estimations of these fractions were made by comparing the area under each peak to that of a known nucleotide having approximately the same Emax and elution position. These data are presented in Table 9. There does not appear to be any specific trend in the amount of either the individual fractions or total nucleotides during growth and development. The total concentrations in mg/40 g of pulp ranged from 0.969 mg to 1.029 mg in 'Orlando' tangelo fruit, 0.779 mg to 1.269 mg in 'Hamlin' fruit, 1.082 mg to 1 .685 mg in 'Pineapple' fruit and O.587 mg to 1.926 mg in 'Parson Brown' fruit. The individual fractions comprising the largest percent of the total amount were a, n, and m. It is also interesting to note that these same fractions were present in all fruits analyzed. Concentration of ATP in Citrus Pulp at Various Stages of Fruit Gro.;lh and Devel opuen t The concentration of ATP in fruit pulp is most often below the

PAGE 117

104 Table 7. Spectroscopic analysis of fractions of a knov/n mixture of 5' mononucleotides separated on a Dov-iex 1 X 8 (formate form) ion-exchange in a formate system Ratio Nucleotide

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105 Table 8, Spectroscopic analysis and tentative identification of the acid-soluble nucleotide fractions extracted from citrus fruit pulp separated on a Dov/ex 1 X 8 (forrivite form) ionexchange in a fornate system

PAGE 119

106 o — — — — — o V

PAGE 120

107 ^ — o — ^ ^ — CnI r— r^ — CSl —

PAGE 121

V 108 minimal level of detection with the UV method of analysis, thus requiring the use of an alternate method such as the 1 uc i fer i n1 uci ferase method described by Buslig and Attaway {2k). These results are presented in Table 10. The most noticeable information that can be obtained from these data is the high ATP concentration in the pulp of young fruit, decreasing with fruit growth and development. The ATP concentrations found in this study are similar to those reported by Bus! ig (23).

PAGE 122

109 Table 10. Concentration of ATP in citrus fruit pulp at various stages of grov;th and development Harvest

PAGE 123

DISCUSSION From the data presented, it can be seen that quantitative changes in nucleotides and R^^!A of the pulp of citrus fruit are gradual from approximately the mid-point of stage II until vjel 1 into stage I I I of fruit development. An attempt will be made in this discussion to relate the concentration of total RNA and of certain RNA species and nucleotides to increases in fruit size, and to juice vesicle content of soluble solids, acids, and proteins. Changes in concentration of total RNA v;ere demonstrated to occur during fruit development ('Orlando' tangelo, 17 nig to Uf mg; 'Hamlin', 82 mg to k? mg; 'Pineapple', 80 mg to 20 mg, 'Parson Brown', 101 mg to 10 mg) . This was true whether total RNA was based on fresh weight or the AISR fraction from fruits. In the case of RNA concentration based on fresh weight, there was a decrease with time as the fruit v/as increasing in size. This basis of comparison does not compensate for an increase in the water content, and growth of the fruit pulp during stage II is primarily by cell enlargement, a process requiring increased water uptake (6). Thus, the apparent decrease in RNA could be the result of increased water content of the system. Indeed, when the increase in fruit volunie became small, RNA content plateaued. In the case of total RNA based on the AISR fraction, an increase occurred at about the same time there was a transition from stage II 1.0 stage I I I of fruit growth of 'Hamlin', 'Pineapple', and 'Parson Brown'. On the other hand, there v/as a slight decrease with 'Orlando' 110

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Ill tangelo. Three points can be made from these comparisons. First, fluctuation in v/ater content of fruits probably v/ould not affect the analyses greatly, and this seerr.sd to be the case. There was no marl
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112 stage II to stage I II of fruit growth. A net increase in protein as a result of increased synthesis did appear to occur during this time since the protein concentration increased as the quantity of AISR decreased. However, part of this increase is possibly only apparent and is due to the loss of pectins from the AISR fraction. The constant level of protein occurring after completion of stage II probably indicates that protein in the fruit at this stage is less susceptible to degradation. Similar findings are reported by Spencer (96). Additional information on the relation of RNA in the fruit during growth and development can be obtained from an analysis of the different species of RNA. Fractionation of RNA can be accomplished several ways; methylated albumen-kiesel guhr (MAK) column, sucrose density gradient, and gel-electrophoresi s. The latter method was chosen because of its quantitative reproducibility and its greater resolution of the various RNA species, parti cularly the ribosomal subunits and lower molecular weight RNAs. Previous fractionation studies of RNA by other workers using gelelectrophoresi s have indicated that RNA separated from green tissue is very different from the RNA separated from non-green tissue (69). Four major' rRNA subunits have been identified in green tissue with sedimentation constants of 25S, 23S, 18S, and 16S. Non-green tissue contained only 2 major rRNA subunits with sedimentation constants of 25s and 18S. The 2 additional components in green tissue, 23S and 18S, have been designated as chloroplast rRNA subunits. The 25S and IBS species are subunits of cytoplasmic rRNA. With the use of the appropriate gel pore size, an additional RNA fraction having a much smaller sedimentation constant can be isolated. This fraction has been des-

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113 ignated as sRNA and is comprised of several RNA species, i.e., kS and 5S. The use of gel -electrophoresi s for the separation of RNA species from citrus fruit pulp tissue yielded excellent, reproducible fractionations. The addition of SDS (0.2% total concentration) to the extraction media and to the electrophoresis buffer was required to prevent any significant RNAase activity. The electrophoretograms of both young and mature fruits were similar to those reported for green and non-green tissue by others. Two major RNA components were common to all fruits analyzed: the 25S and 18S subunits of cytoplasmic rRNA and sRNA. However, the electrophoretograms of young fruit differed from those of mature fruit in 2 respects. First, young fruit in the early part of stage II of growth contained 2 minor RNA components, Rf's 0.28 and O.k], which were absent from the electrophoretograms of mature fruit; and secondly, the quantity of each component was greater in young fruit than in mature fruit. The 2 minor components are of particular interest because they correspond to the chloroplastic rRNA subunits reported by others (^7, 69, 103). Yet, the fruit vesicles are devoid of chloroplasts; however, chromoplasts are present (89). Possibly, these 2 minor components are present in all plastids. The loss of these 2 components with growth is probably the result of degradation. Ingle (U7) , studying the synthesis and stability of chloroplastic rRNA, found that as the chloroplasts matured the percent degradation also increased. There was also a decrease in quantity of each RNA component with fruit growth of all k varieties. Studies by Ismail (50) and others (27, 78, 89, 91) have reported similar findings in other types of tissue, but they

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reported an increase in the sRNA that accompanied the decrease in rRNA. This was especially noticeable in tissue during periods of rapid degradation of ribosomal RNA. However, it appears from this study that rRNA degradation does not contribute to sRNA. Further analysis of the RNA species can be made by measuring the area under each peak and expressing the value as a ratio of the subunits for each rRNA. These ratios have been used by others to indicate the stability and turnover of rRNA (^+7, 103). The ratio of the cytoplasmic rRNA subunits, 25S and 18S, is usually 1.8-2.0 in most plant tissues (104). This is typical of cotyledons of seedlings 5 to 6 days after germination (103), and is often characteristic of plant tissue in a resting state (69). Increases in either rRNA synthesis or degradation or both, are thought to be reflected in ratios deviating from 1 .8-2.0. The 25S:l8S ratio of the citrus fruit pulp increased up to the completion of stage II of fruit growth. Based on the study of Ingle (^7) , it would seem that the stability of the cytoplasmic rRNA of citrus increases up to the completion of stage II and remains stable well into stage III. This is further supported by the increase in the rRNA:sRNA ratio during the same period. Ismail (50) and others have found that with drastic declines in rRNA there was substantial increase in sRNA. The low ratios of 1.0 to 1.4 found in enlarging fruits (collected July 23, 1970, and September 26, 1970), is apparently the results of an active turnover in cytoplasmic rRNA. Increased cell division of the juice vesicle is occurring at this time (7). 32 The synthesis of RNA can be studied with the use of P incorporated into RNA, separation of RNA by gel -e lectrophores i s , and detection of ^ P incorporation in the various RNA compounds. Studies of this

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115 type v;ith ' Caloi.iondl n ' fruits yielded data similar to those reported by others (50, 68, 8k). ^'^-p activity v^as found in the RNA components of the electrophoretogranis of both young and mature, green fruit pulp, but absent in tiie electrophoretograms of very mature fruit prior to 32 abscission. The amount of ' P incorporated into the P.MA of mature, green fruit v/as greater than that i ncoi'porated by young, enlarging fruit, indicating that the rate of RI^IA synthesis was greater in the former fruits. These data further support the increased RNA level found in the AISR fraction (Table k) of the other varieties at completion of stage II of fruit gro\7th since the mature green 'Calamondin' fruits probably correspond in development to the latter portion of stage II. Particularly interesting was the amount of ^^P activity in the region of mRIJA (RF 0.50) of the electrophoretogram of mature, green 'Calamondin' fruit. Several workers have designated this species as being a DNA-RNA complex {kS, kS , 50). It Is widely accepted that this DNA-RMA complex Is an Intermediate In the transcription of genetic information. It is suggested that synthesis of this complex In 'Calam.ondin' , and possibly otiier citrus fruits, is one of the basic metabolic changes necessar-y to initiate fruit ripening and eventual senescence. Richmond and Biale (8^+) studying avocado fruit found that 32 P Incorporation into rRMA and mRfIA was high in tlic early phases of the cl Itnacterlc, declined v/itii the advent of the greatly increased utilization of 0^, and v;as absent at the maximum point of respiration. Frcni
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116 sis necessary for all subsequent proteins synthesized and required for ripening. Ethylene has been found to hasten fruit ripening (21, 22) and also has been reported to cause a synthesis of the DfJA-RNA complex (1, 2, 50). Thus these data support the idea of an increase in niRNA, and possibly protein synthesis just prior to the physiological events associated with senescence. The electrophoretograms of very mature 'Calamondin' fruit prior to abscission showed an absence of distinct RNA components. This indicates much reduced synthesis of RNA. Similar data have been reported by Ismail (50). It v/ould seem that maintenance of RNA synthesis well into stage Ml is a very important characteristic vjhich distinguishes citrus fruit, a non-climacteric fruit, from climacteric fruits. Several studies have indicated that degeneration of cells is accompanied by a failure to synthesize RNA (50, 63, Sk) . Ismail (50) was able to delay senescence in 'Calamondin' fruit peel and maintain synthesis of RNA v;ith GA applications. Application of Kinetin, a senescence retardant, showed similar effects (62, 79, 97). Leopold (67) has suggested that senescence is regulated by RNA„ In contrast, ethylene treatments are reported to accelerate senescence in citrus fruits (39, 50) and other tissues (20), and cause a concommitant degradation of RNA (50). Recently Ku and Romani (63), using pear fruit, found that the onset of senescence was associated v;ith a rapidly decreasing rate of ribosomal turnover, leading to a cessation of rRNA synthesis as the fruit reached the climacteric peak. In the same study, a distinct loss in polysomes occurred only in the post-climacteric phase. Similar findings for avocado fruits

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117 are reported by Richmond and Biale (3A) . The burst of ethylene associated with ripening of climacteric fruits must greatly contribute to increased RMA metabolism observed in these fruits. It is interesting to note that removal of ethylene from tissue during the climacteric period, or preventing its production, delays ripening and the rate of cellular degradation (22). The most important information gained from these data is that RNA in the fruit pulp does not undergo drastic changes such as observed during seed germination (27, 73) and during the climacteric phase of apples (70), pears (63), and avocadoes (8^). Tissue culture studies of the juice vesicles by Bell (11) also support these findings. He observed that cells of the juice vesicles from mature and immature fruits, cultured in vitro differentiated into distinct juice vesicles and it was difficult to stimulate the tissues to produce callus-lil
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118 compounds at the beginning of gradient would alter the absorption spectrum of those nucleotides being eluted simultaneously. NAD is reported to be eluted from a formate an ion-exchange colunn simultar neously v;ith CMP (a) and AMP (b) (18). Also, the elution of GDP simultaneously with CTP and UDP simultaneously with ATP was found to occur in the standards. Peaks n and o (Fig. 30 could possibly be comprised of GDP-CTP and UDP-ATP, respectively. The identity of the fraction at position m is not known. Although, it v/as present in all samples analyzed and at a relatively high concentration. The only nucleotide positively identified and quantitated was ATP. Determinations of this nucleotide \-jere mads by the 1 uci fer i n1 uci ferase procedure specific for ATP {2k). The ATP concentrations in these various fruits are in agreement with those reported by Buslig (23) and would comprise less than 3% of the total concentration of peak o. It is difficult to suggest particular roles for these compounds in the metabolism of the fruit pulp since conclusive identification has not been established. Yet, the quantity of these compounds found in the pulp and their tentative identification does suggest they are important intermediates in metabolism, particularly carbohydrate m.etabolism. NAD and the other nucleotides tentatively identified in these extracts are essential intermediates in both glycolysis and the organic acid cycle (29). Hassid (^3) has discussed in detail the significance of nucleotides in the transformation of sugars in plants. Recently, Buslig (23) suggested that ATP is important in controlling acid accumulation in citrus fruit. ATP may be required for the translocation of acid from the r, i tochondr ia to the vacuole. A non-synchrony of syntliesis of enzymes associated with the cittic acid cycle gives

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119 rise in the mitochondria to increased levels of citric acid. in the present study, the adenosine nucleotides were abundant in young fruits, coinciding vvith tine of accumulation of acid in the vesicles, and declined to a very lovi level in mature fruits. A number of studies have recently been made on nucleotides and their influence on modifying flavor (6^+, 65, 92). These studies indicate that the 5' mononucleotides vjere much stronger modifiers of flavor than the other derivatives. Of the 5' mononucleotides generally found in plant tissue, 5' GMP probably has the strongest effect. The minimum concentration of these compounds required to modify flavor is only several ppm. The concentration of several nucleotides in juice vesicles v.'as in excess of this amount.

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SUMMARY AND CONCLUSION The quantity of nucleotides and RNA of citrus fruit during grov/th and development were studied in relation to increases in fruit size and to soluble solids, acidity, and proteins. Experimental material consisted of fruits of 'Orlando' tangelo, and 'Hamlin', 'Pineapple', and 'Parson Brown' oranges. It was concluded from this study that quantitative changes in RNA are gradual over the stages of fruit development examined. This conclusion was based on the following observations from total RNA and gel -e lect rophoret i c determinations: Changes in concentration of total RNA were gradual from the mid-point of stage II until well into stage III, with only a slight increase just prior to completion of stage II of fruit growth. Gel -elect rophoret i c separation of RNA yielded patterns that indicated that RNA changes were gradual decreasing until the transition from stage II to stage III and then remained fairly constant for at least 2 months, and possibly longer. 32 In 'Calamondin' fruit, maximum incorporation of P into RNA occurred also at the transition from stage II to stage III, and appreciable ' P activity was found in the mRNA region of the e lect rophoretogram. This component Is believed to be a DNA-RNA complex. It was suggested that the increased RNA in the early part of stage II I of fruit development may be essential for the initiation of changes associated with ripening of citrus fruit, e.g., production of enzymes required for changes normally associated with ripening. 120

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121 Analysis of the acid-soluble nucleotide fraction of the fruit indicated that they are present in appreciable quantities in fruits and most surely play a vital role in fruit development. The nucleotides NAD, CMP, AMP, UMP, ADP, CTP, GDP, UDP, and ATP were tentatively identified based on the elution position from an an ion-exchange column and spectral analysis. ATP was further assayed by the luciferinluciferase method. The possible significance of nucleotides in carbohydrate metabolism and in the edible fruit v;as discussed, particularly as nucleotides may modify flavor.

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LITERATURE CITED 1. Abeles, F. B. 1966. Auxin stimulation of ethylene evolution. Pit. Physiol, k] :585-588. 2. and R. E. Holms. 1966. Evidence for hormonal role of ethylene in abscission. Pit. Physiol. Suppl . ^1 : 1 i i i , 3. Attardi, G. and F. Amaldi. 1970. Structure and synthesis of ribosomal RNA. Ann. Rev. Biochem. 39:183-225. k. Axelrod, B. 19^7. Citrus fruit phosphatases. J. Biol. Chem. 167:57-72. 5. , R. Jang, and J. M. Lawerence. 1955. Glutamic acid decarboxylase of lemons and oranges. J. Ag. Food Chem. 3: 1039-lOi+l. 6. Bain, Joan M. 1958, Morphological, anatomical, and physiological changes in the developing fruit of 'Valencia' orange, Ci trus sinensis (L.) Osbeck. Aust. J. Bot. 6:1-24. 7. Baker, R. A. and J, H, Bruemmer, 1969. Cloud stability in the absence of various orange juice soluble components. Amer. Soc. Hort. Sci. 82:215-220. 8. Barthalomevy, E. T. and H. S. Reed. 19^3. General morphology, hibtology, and physiology. _l_n The Citrus industry. Weber, H. J. and L. D. Batchelor (eds.). 1:669-717. Univ. of Calif. Press, Berkeley. 9. and W. B. Sinclair, 19^3, Soluble constituents and buffer properties of orange juice. Pit. Physiol, 18:185-206, 10. Beevers, L, 1966, Effect of gibberellic acid on the senescence of leaf disc of Nasturtium (Tropaeolum majus ) , Pit. Physiol, 41 : 10741076. 11. Bell, D. 1971. Personal communication. Univ. of Florida, Gai nesv ! 1 le. 12. Biale, J. B. I960. Respiration of fruits. J_n Handbuch der Pflanzenphysiologia. Ruhland, W. (ed. ). XI 1/ I I :536-592. Spr i nger-Verlag, Berlin, 22

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123 13. • 1961. Postharvest physiology and chemistry. Jjn The Orange. Sinclair. W. B. (ed.). pp. 96-130. Univ. of Calif. Press, Berkeley. 14. and R. E. Young. 1962. The biochemistry of fruit maturation. Endeavor 21:164-174. 15. Braverman, J. S. 1933. The chemical composition of the orange. Hadar 6:62-65. 16. Bonner, D. M. I96O. Control Mechanisms In Cellular Processes. The Ronald Press, New York. 17. Bonner, J. and J. E. Varner. I965. Plant Biochemistry. Academic Press, New York. 18. Brov;n, E, G. 1962. The acid-soluble nucleotides of mature pea seeds. Biochem. J. 85:633-640. 19. Brown, J. and P. Nordin. I969. Soluble nucleotides from the immature fruit of tomato. J. Ag. Food Chem. 17:341-343. 20. Burg, S. P. I968. Ethylene, plant senescence and abscission. Pit. Physiol. 43:1503-1511. 21. and E. A. Burg. 1962. Role of ethylene in fruit ripening. Pit. Physiol. 37:179-189. 22. and . I965. Ethylene action and the ripening of fruits. Science 148:1190-1196. 23. Buslig, S. B. 1970. Biochemical basis of acidity in citrus fruits. Doctoral Diss., Univ. of Fla., Gainesville. 24. and J. A. Attaway. I969. A study of acidity levels and adenosine triphosphate concentration in various citrus fruits, Proc. Fla. State Hort. Soc. 82:206-208. 25. Chandra, G. R. and J. E. Varner. I965, Gibbereilic acid-controlled metabolism of RNA in aleurone cells of barley. Biochem. et, Biophys. Acta. 108:583-592. 26. Chantreen, H. I96I. The Biosynthesis of Protein. Pergaman Press, Oxford. 27. Cherry, J. H., H, Chraboczek, and W, J. G. Carpenter. I966. Nucleic acid metabolism in peanut cotyledons. Pit. Physiol, 40:582-587. 28. Clements, R. L. and H. V. Leland. 1962. Seasonal changes in the free amino acids in 'Valencia' orange juice. Amer. Soc. Hort. Sci. 80:300-307.

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]2k 29. Conn, E. C, and P. K. Stumpf. I966. Outlines of Biochemistry. 2nd ed, John V/iley and Sons, Inc., New York 30. Davis, W. B. 19^^2. The di sti'i but ion and preparation of citrus peroxidase. Amer. J. Bot. 29:252-25'f, 31. Dosta] , H. C. 1970. The biochemistry and physiology of ripening. Hort. Science 5:36-37. 32. Fan, D, F. and G. A. Maclachlan. I967. Massive synthesis of ribonucleic acid and cellulase in the pea epicotyl in response to indoleacetic acid, v;ith and v/ithout concurrent cell division. Pit. Physiol. h2:]]]k-]]22. 33. Fletcher, Px. A, and D. J. Osborne. I965. Regulation of protein and nucleic acid synthesis by gibberellin during leaf senescence Nature 207:1 176-1177. 3^. and . 1966. GA as a regulator of protein and RNA synthesis during senescence in leaf cells of Taraxacium officinale . Can. J. Bot. 44:739-7^7. 35. Fox, J. E. 1964. Incorporation of kinin into the RNA of plant tissue cultures. Pit. Physiol. Suppl . 39:xxxi. 36. . 1966. Incorporation of a kinin, N -benzyladeni ne, into soluble RNA. Pit. Physiol. 41:75-82. 37. Frenkel, C. , 1. Klein, and D. R. Oilley. I96S. Protein synthesis in relation to ripening of pome fruits. Pit. Physiol. 43: 1146-1153. 38. Gates, C. T. and J, Bonner. 1959. The response of the young tomato plant to a brief period of water shortage. IV. Effects of viater stress on the ribonucleic acid metabolism of tomato leaves. Pit. Physiol. 34:49-55. 39. Grierson, V/. and V/. F. Newhall. I96O. Degreening of Florida citrus fruits. Univ. of FK-i. Ag. Exp. Stat. Bui. 620, p. 79. 40. Hamilton, T. K. , R. H. Moore, A. F. Ruinsey, A. R, Moans, and A. R. Schrank. I965. Stimulation of syiithesis of ribonucleic acid in sub-apical sections of Avena coleoptile by indoly-3acetic acid. Nature 208: 1 IBO1 1 83 . 41. Hansen, E. I96S. Postharvest physiology of fn.iits. Ann. Rev. Pit. Physiol. 17:459-480. 42. Harding, P. L., J. R. Winston and D. F. Fisher. 1940. Seasonal changes in Florida oranges. Tech. Bui. No. 753. USDA.

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125 ^3. Hassid, W. Z. 1967Transformation of sugars in plants. Ann, Rev. Pit. Physiol . 18:253-280. hk . Hayasini, M. and S. Speigelman. I96I. The selective synthesis of informational RMA in bacteria. Proc. Matl. Acad. Sci., U.S. ij7: IBS'; -1580. 45. Holdgate, D. D. and T. W. Goodv/in. 1965. Quantitative extraction and estimation of plant nucleic acids. Phytochem. •'<:831-8^3. hG. Hulme, A. C. 195'*. Studies in the nitrogen metabolism of apple fruits. The climacteric rise in respiration in relation to changes in the equilibrium between protein synthesis and degradation. J. Exptl. Bot . 5:159-172. hj . Ingle, J. I968. Synthesis and stability of chloroplast ribosomalRNAs. Pit. Physiol. k3:1hh8-]h5h . ^8. and J. L. Key. 1965. A comparative evaluation of the synthesis of DNA-like RIJA in excised and Intact plant tissue. Pit. Physiol . ^10:1212-1219. kS. , , and R. E, Holm. I965. Demonstration and characterization of a DMA-like RtJA in excised plant tissue. J. Moll. Biol. 11:730-7^6. 50. Ismail, M. A. 1966. Polymeric nucleic acids from citrus fruits and leaves in various stages of grov/th and development, including chemically modified senescence. Doctoral Diss., Univ. of Fla . , Gainesvi lie. 51. Jensen, E. F. , R, Jang, and J. Bonner. I960. Orange pectlnesterase binding and its activity. Food Res. 25:6^-72. 52. , , and L. R. MacDonnell. 19'(7. Citrus acetylesterase. Arch. Biochem. 15 : 'il5-'i31 • 53. Jansen, V/. A. 1957. The incorporation of 'c-adenine and Cphcny ladan ine by developing root tip cells. Proc. Matl. Acad. Sci. U.S. k3:103Q-10he. 5^. Kefford, J. F. and B. V. Chandler. 1970. The chemical constituents of citrus fruits. J_n^ Advances in Food Research. Chichester, C. 0., E. M. Mrak, and G. F. Stewart (eds.). Academic Press, New York. 55. Kcssler, B. and M. Engelberg. I962. Ribonucleic acid and ribonuclcase activity in developing leaves. Biochem. et. Biophys. Acta. 55:70-32. 56. Key, J. L. 196't. Ribonucleic acid and protein synthesis as essenLial for cell elongation. Pit. Physiol. 39:365-370.

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126 57. • 1969. Hormones and nucleic acid metabolism. Ann. Rev. Pft. Physiol. lO'.kkS-ky^ . 58, . 1971. Auxin-regulated RHA synti^esis: Present status and anticipated approaches. _l_n Whats Uevi in Plant Physiol. ^ 3(9):l-5. Fritz, G. (ed,). Univ. of Fla., Gainesville. 59. and J, Ingle, I968. RMA metabolism in response to auxin. _[n Biochemistry and Physiology of Plant Grov/th Substances. Wightman, F. and G. Setterfield (eds,). pp. 711-722. The Runge Press Ltd., Ottawa, Canada, 60. and C. Y, Lin, I966. Relation of 2,4-D induced grovjth aberrations to changes in nucleic acid metabolism in soybean seedlings. Bot. Gaz. 127:87-9^. 61. and J, C, Shannon, 19^'+. Enhancement by auxin of MiA synthesis in excised soybean hypocotyl tissue. Pit. Physiol . 39:360-36^+. 62. Krul, V/. R, and A. C. Leopold. I965. The role of nucleic acids in leaf senescence. Pit. Physiol, Suppl . 40 : 1 i . 63. Ku, L. L, and R, J, Romani, 1970, The ribosomes of pear fruit. Pit. Physiol . ^5:^+01-^07. 6k. Kuninaka, A. I96O. Studies on taste of ribonucleic acid derivatives. J, Agri, Chem. Soc, Japan 3^:^89. 65. , M. Kibi, and K. Sakaguchi. 196'-f. History and development of flavor nucleatides. Food Tech. 18:287-293. 66. Leaver, C. J. and J. L, Key. 1970, Ribosomal synthesis in plants. J. Mol. Biol, i+9:671-680. 67. Leopold, A. C. 196'+. Plant Growth and Development. McGrawHill Book Company, p, I96. New York. 68. Loening, U, E. 1967. The fractionation of high m.olecular v/eight ribonucleic acid by pol yacrylamide-gel electrophoresis, Biochem. J. 102:251-257. 69. and J. Ingle. I967. Diversity of RfIA components in green plant tissue. Nature 215:363-367. 70. Looney, N, E. and M. E. Patterson. I967. Changes in total ribonucleic acid during the climacteric phase in ycllov; transparent apples, Phytochem, 6:1517-1520. 71. HacDonn:;]!, I.. R., R. Jang, E, F. Jansen, and H, Uneweaver. 1950. The specificity of pecti n-esterase from several sources v/ith notes on purification of orange pecti nesterase. Arch, Biochem, 28:260-273.

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127 72. Mazur, A. and B. Harrow. 1969. Biochemistry: A Brief Course. W. B. Saunders Company. Philadelphia, Pa. 73. Melera, P. W. 1971. Nucleic acid metabolism in germinating onion. Pit. Physiol. it8:73-8l. 7k. Nitsan, J. and A. Lang. 1966. DNA synthesis in the nondividing cells of the lentil epicotyl and its promotion by gibberellin. Pit. Physiol. 41:965-970. 75. Nitsch, J. P. 1965. Physiology of flower and fruit development. In Handbuch der Pf lanzenphysiologia. Ruhland, W. (ed.). XV/l :1537-1627. Springer-Verlag, Berlin. 76. Pgur, M. and G. Rosen. 1950. The nucleic acids of plant material. I. The extraction and estimation of desoxypentose nucleic acids and pentose nucleic acids. Arch. Biochem. 25:262-267. 77. Oota, Y. 196^+. RNA in developing plant cells. Ann. Rev. Pit, Physiol. 15:17-36. 78. and K. Takata. 1959. Changes in microsomal ribonucleoprotein in the time course of germination. Physiol. Planta. 12:518-525. 79. Osborne, D. J. 1962. Effect of Kinetin on protein and nucleic acid metabolism i n Xanth i urn leaves during senescence. Pit. Physiol. 37:595-602. 80. Paleg, L. G. I96O. Physiological effects of gibberellic acid. I. Carbohydrate metabolism and amylase activity of barley endosperm. Pit. Physiol. 35:293-299. 81. , 1965. Physiological effects of gi bberel 1 i ns, Ann. Rev. Pit. Physiol. 16:291-322. 82. Potty, V. H. 1969. Determination of proteins in the presence of phenols and pectins. Anal. Biochem. 29:535-539. 83. Rasmussen, G. K. 1964. Seasonal changes in the organic acid content of 'Valencia' orange fruit in Florida. Amer. Soc. Hort. Sci. 84:181-187. 84. Richmond, A. and J. B. Biale. 1967. Protein and nucleic acid metabolism in fruits. II. RNA synthesis during the respiratory rise in the avocado. Biochim. Biophys. Acta. 138:625-627. 85. Robinson, E, and R. Brown. 1954. Enzyme changes in relation to cell growth in excised root tissue. J. Exptl. Bot. 5:71-78. 86. Rockland, L. B. 1964. Nitrogenous constituents. J_n The Orange. Sinclair, W. B. (ed.). pp. 230-264. Univ. of Calif. Press, Berkeley.

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128 87. Rouse, A. H. , C. D. Adkins, and E. I. Moore. 1962. Seasonal changes occurring in the pecti nesterase activity and pectin constituents of component parts of citrus fruits. I. 'Valencia' oranges. J. Food Sci. 27:^+19-425. 88. Sacher, J. A. I968. Senescence: effects of auxin and Kinetin on RNA and protein synthesis in subcellular fractions of fruit and leaf tissue sections. J_n Biochemistry and Physiology of Plant Growth Regulators. Wrightman, F. and G. Setterfield (ads.), pp. 1457-1^78. The Runge Press Ltd., Ottawa, Canada. 89. Schneider, H. 1968. The anatomy of citrus. _[n The Citrus Industry. Reuther, W. , L. D. Batchelor, and H. J. Weber (eds.). 11:1-22. Univ. of Calif. Press, Berkeley. 90. Selvendran, R. R. and F. A. Isherwood. I967. Identification of guanosine diphosphate derivatives of D-xylose, D-mannose, D-glucose, and D-galactose in mature strawberry leaves. Biochem. J. 105:723-728. 91. Shaw, M., P, K. Bhttacharya, and W. A. Wuick. I965. Chlorophyll, protein, and nucleic acid levels in detached senescing wheat leaves. Can. J. Bot. ^+3:739-7^. 92. Shimazono, H. I96U. Distribution of 5' ribonucleotides in foods and their application to foods. Food Tech. 18:29^-303. 93. Silberger, J. and F. Skoog. 1953. lAA induced changes in nucleic acid content and growth of excised tobacco pith tissue. Science ]]8:kk3-kkh. Sk. Sinclair, W. B. I96I. Principal juice constituents. In The Orange. Sinclair, W. B. (ed.) . pp. I3I-I6O. Univ. of Calif. Press, Berkeley. 95. Smillie, R. M. and G. Krotkov. 1959. Ribonucleic acids as an index of metabolic activity of pea leaves. Biochem. et. Biophys. Acta. 35:550-554. 96. Spencer, Mary. I965. Fruit ripening. J_n Plant Biochemistry. Bonner, J. and J. E. Varner (eds.). Academic Press, New York. 97. Srivastava, B., I. Sahi, and G. Ware. I965. The effects of Kinetin on nucleic acids and nucleases of excised barley leaves. Pit. Physiol. 40:62-64. 98. Stewart, J. McD. and G. Guinn. 1971. Chilling injury and nucleotide changes in young cotton plants. Pit. Physiol. 48:166-170.

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129 99. Sturani, E. and S. Cocucci. 1965. Changes in the RMA system in the endosperm of ripening castor bean seeds. Life Sciences ^: 1937-19^'+. 100. Thomas, A. J. and N, S, A. Sherratt. 1956. The isolation of nucleic acid fractions from plant leaves and their purine and pyrimidine composition. Biochem. J. 62:1-^. 101. Vorma, T. N. S. and C. V . Ramakrishan. 1956. Biosynthesis of citric acid in citrus fruits. Nature 178:1358-1359. 102. Varner, J. and G. R. Chandra. 196^4. Hormonal control of enzyme synthesis in barley endosperm. Proc, Natl. Acad. Sci., U.S. 52:100-106. 103. Vedel, F. and M. J. D'Aoust. 1970. Pol yacry 1 ami de gel analysis of high molecular v/eight ribonucleic acid from etiolated and green cucumber cotyledons. Pit. Physiol. ^(6:81-85. 104. and . 1970. Rapid separation of ribosoma] RNA by sucrose density gradient centr i f ugat ion with a fixed angle rotor. Anal. Biochem. 35:5'^-59. 105. Vines, V. M. I968. Citrus enzymes. 11. Mitochondria and cytoplasmic malic dehydrogenase from grapefruit juice vesicles. Amsr. Soc. Hort. Sci. 92:179-18^^. 106. V/einstein, L. H., D. C. McCune, J. F. Mancini, and P. Van Leuken. 1969. Acid soluble nucleotides of pinto bean leaves at different stages of development. Pit. Physiol. ^';: 1 't991 5I 0. 107. V/est, S. j-i. 1962. Protein, nucleotide, and ribonucleic acid metabolism in corn during germination under water stress. Pit. Physiol . 37:565-567. 108. , J. B. Hanson, and J. L. Key. 1950, Effects of 2,^1-0 on nucleic acid and protein content of seedling tissue. Weeds 8:333-3^fO.

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BIOGRAPHICAL SKETCH Charles Rice Barmore was born December 26, 19^2, in Leesburg, Florida. In June, 19^1, he graduated from Grovejand High School. In December, 1966, he received the degree of Bachelor of Science in Dairy Science from Clemson University. After graduation, he v/as employed as a research technician v;ith the United States Department of Agriculture in Orlando, Florida, In September, 1967, he enrolled in the Graduate School of the University of Florida. He v;orked as a graduate research assistant in the Department of Fruit Crops until March, 1972. In March, 1969, he received the degree of Master of Science in Agriculture, From March, 1969, until the present time he has pursued his vjork tov;ard the degree of Doctor of Philosophy. The author is a member of Sigma Xi , Gamma Sigma Delta and Alpha Zeta honorary fraternities. 130

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I certify that I have read this study and that in my opinion it conforms to accpntable standards of scholarly orpsenfation ^nd i =; full' adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Revert H. Professor Department Fruit Crops I certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Professor and C Department i certify that 1 have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. sJ4ifma^ ^* ^ (pU/juJC) Thomas E. Humphreys Professor and Biochemist, Botany Department I certify that I have read this study and that In my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Smi th Assistant Professor of Botany, Botany Department

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholar!/ presentation and Is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. H/ill Fam J. Wi Itbank Assistant Professor (Assistant Hort culturist), Fruit Crops Department This dissertation was submitted to the Dean of the College of Agricul-* ture and to the Graduate Council, and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. March, 1972 Dean, Graduate School

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