NITROGEN FIXATIDN AND CARBOHYDRATE PARTITIONING IN PHASEDLUS VULGARIS L. Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY ROMEO MARTINEZ RODAS - 1976 Ilid III III II I III . .12: v , .ate University This is to certify that the thesis entitled NITROGEN FIXATION AND CARBOHYDRATE PARTITIONING IN PHASEOLUS VULGARIS L. presented by Romeo Mar t ine z-Rodas has been accepted towards fulfillment of the requirements for Ph.D. degree in Crop Science M. W. Adams Major professor Date February 25, 1977 0-7639 ABSTRACT NITROGEN FIXATION AND CARBOHYDRATE PARTITIONING IN PHASEOLUS VULGARIS L. BY Romeo Martinez Rodas The ontogenetic relationships that may exist between symbiotic nitrogen fixation and carbohydrate partitioning were studied in four dry bean cultivars. Total soluble carbohydrate and starch contents of the primary nodule population were found to be closely correlated in time with nitrogen fixation activity. The decline in nitrogen fixation activity was found to be correlated with loss of nodule dry weight, decreased total soluble carbohydrate and starch of nodules and an increase in soluble carbohydrate and starch in leaves and stems during the early stages of reproductive growth. Starch concentration was found to decline one week prior to the occurrence of nitrogen fixation maxima suggesting that hydrolysis of this polysaccharide may contribute energy to the fixation of molecular nitrogen. Temporary sites of starch accumulation were found to be restricted primarily to parenchyma cells of secondary xylem and pith and to uninfected cells in nodules. Reduction of carbohydrate movement to the nodule, due to "competition" by reproductive sinks, may not be the cause of the observed decline in nitrogen fixation. It is suggested that a irrelopee is abser 1152 i i: went ‘ I XIII SCSI Romeo Martinez Rodas developmentally compensated genetic program predisposes the nodules to the observed decline in N2 fixation. Thus, loss of nodule competency in N2 fixation may not be triggered by a reduction in carbohydrate movement to such structures but by an activating signal(s) affecting both nodule and lower leaf senescence, probably hormonal in nature. Dry bean plants exposed to 1200 ppm carbon dioxide at various developmental stages exhibited higher nitrogen fixation rates, higher nodule fresh weights, higher total soluble carbohydrate and slightly higher organic nitrogen contents. Carbon dioxide treatment during the four weeks prior to reproductive growth was found not to extend the duration nor prevent the decline of nitrogen fixation. The view that the rate of nitrogen fixation pg; se is limited by photosynthate available to the entire symbiotic system is supported. NITROGEN FIXATION AND CARBOHYDRATE PARTITIONING IN PHASEOLUS VULGARIS L. by Romeo Martinez Rodas A DISSERATION Submitted to Michigan State Univeristy in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1976 .I , can- P ant! . ~.F..n . HA I An] . ..““-u v . ‘gux we? t» .A “4: 3:; Y L ACKNOWLEDGEMENTS For the freedom and encouragement given me at Michigan State University in sharing ideas and in exploring and honestly confronting varying points of view, I wish to express my sincere appreciation to Dr. Maurice Wayne Adams, Dr. John E. Grafius, Dr. Peter H. Graham (CIAT), Dr. Donald Penner, and Dr. James M. Tiedje, members of my Guidance Committee. Specialthanks are extended to the Centro Internacional de Agricultura Tropical (CIAT) in Cali, Colombia, and to CIAT staff members, including particularly, Dr. Peter H. Graham and members of the Bean Research Group with whom I had the privilege of working for one year during the research phase of my doctoral program. I wish to acknowledge with great appreciation the financial support of the Rockefeller Foundation which made my graduate education possible, and to extend my personal thanks to Drs. Robert K. Waugh and L. M. Roberts of the Foundation for their interest and efforts in my behalf. I am most deeply grateful to my wife, Lucrecia, and our children for their constancy, support and encouragement throughout the extended period of dislocation attendant to the study, travel and research required in my doctorate program. ii I'~ “'1 -.r L. ‘*" ~n..\ “IN- I-' 'h.‘ ‘ “A 'l ~-x TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . xii INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . 1 CHAPTER 1: SYMBIOTIC NITROGEN FIXATION . . . . . . . . . . . . 3 Introduction. . . . . . . . . . . . . . . . . . . . . . 3 Ontogenetic Nitrogen Fixation . . . . . . . . . . . . . 4 References. . . . . . . . . . . . . . . . . . . . . . . 14 CHAPTER 2: CARBOHYDRATE PARTITIONING AND NITROGEN FIXATION IN PHASEOLUS VULGARIS L. . . . . . . . . . . . . . . . 17 Abstract. . . . . . . . . . . . . . . . . . . . . . . . 17 Introduction. . . . . . . . . . . . . . . . . . . . . . 18 Materials and Methods . . . . . . . . . . . . . . . . . 18 Results and Discussion. . . . . . . . . . . . . . . . . 22 References. . . . . . . . . . . . . . . . . . . . . . . 67 CHAPTER 3: EFFECTS OF CARBON DIOXIDE ENRICHMENT ON SYMBIOTIC NITROGEN FIXATION IN PHASEOLUS VULGARIS L.. . . . . 69 Abstract. . . . . . . . . . . . . . . . . . . . . . . . 69 Introduction. . . . . . . . . . . . . . . . . . . . . . 70 Materials and Methods . . . . . . . . . . . . . . . . . 70 Results and Discussion. . . . . . . . . . . . . . . . . 74 References. . . . . . . . . . . . . . . . . . . . . . . 35 CHAPTER 4: SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 86 SELECTED BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 90 APENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 iii rt; LIST OF TABLES Page 11. Dinitrogen fixed and percentage of total nitrogen fixed of ten sequential weekly intervals of four dry bean cultivars . . . . . . . . . . . . . . . . . 26 2. Cubic polynomials fitted to stem dry weights (A) and leaf dry weights (B) and harvests (t, independent variable) of four dry bean cultivars (C). First and second derivatives, maximums, and points of inflection are shown. . . . . . . . . . . . . . . . . . . . . . 35 3. Linear regression equations predicting nitrogen fixation rates for a given level of nodule soluble carbohydrate, and a given level of nodule starch . . 46 4. Organic nitrogen content of vegetative structures (roots, stems, and leaves) of ten sequential weekly harvests of four dry bean cultivers. Analysis of Variance is presented in Appendix Table 44. . . . . SS 5. Cubic polynomials fitted to organic nitrogen content of stems (A), leaves (B) and, harvests (t independent variable) of four dry bean cultivars. Corresponding 95% confidence upper and lower limits (CL) are given. . . . . . . . . . . . . . . . . . . . . . . . 56 6. Effect of carbon dioxide on nitrogen fixation rates and nodule fresh weight of the dry bean cultivar Porrillo Sintetico . . . . . . . . . . . . . . . . . 75 7. Effect of carbon dioxide on root fresh weight, stem fresh weight, leaf fresh weight, and pod fresh weight of the dry bean cultivar Porrillo Sintetico. . . . . 76 8. Effect of carbon dioxide on root dry weight, stem dry weight, leaf dry weight, and pod dry weight of the dry bean cultivar Porrillo Sintetico . . . . . . 78 9. Effect of carbon dioxide on content and concentration of soluble carbohydrates of nodules, roots, and stems, of the dry bean cultivar Porrillo Sintetico. . . . . 79 10. Effect of carbon dioxide on content and concentration of organic nitrogen of roots, stems, leaves, and pods of the dry bean cultivar Porrillo Sintetico . . . . . . 81 iv Ill. Effect of carbon dioxidecnlcomponents and subcomponents of yield of the dry bean variety Porrillo Sintetico at maturity . . . . . . . . . . . . . . . . . APPENDICES l. 43. 10. Dry weights (in grams/15 plants) of primary roots of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Dry weights of "secondary roots" (in grams/15 plants) of ten sequential weekly harvests of four dry bean Cultivars, and corresponding Analysis of Variance. Cubic polynomials fitted to stem dry weight (dependent variable) of four dry bean cultivars, and corresponding Analysis of Variance . . . Dry weights of leaves (in grams lS/plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Anaylsis of Variance . . . . . . Cubic polynomials fitted to leaf dry weight (dependent variable) of four cultivars, and corresponding Analysis of Variance. . . . . . . . . . . . . . . . . . . . . Dry weights of vegetative structures (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Dry weights of flowers and rachis (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Dry weight of pods (in grams/15 plants) of ten sequential weekly harvests of four dry bean cultivars. and corresponding Analysis of Variance . . . . . . . . Dry weights of reproductive structures (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. . . . Total dry weights of plants (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . Nitrogen fixation rates (u moles CZHA Spl-lhr-l) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. . . Page 83 95 96 97 99 100 102 103 104 105 106 107 ll. 12. 13. IS. 16. 19. 21. NM seq- cul Tot nod hat por no: WI (:0 To of cu nmnH (fir-nan CQ'OH OC'UC) nmnn 114 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Nodule dry weights (in grams/15 plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Total content of ethanol soluble carbohydrates in nodules (in mg/lS plants) of eight sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance . . . . . . . . Concentration of ethanol soluble carbohydrates in nodules (in mg/g nodule dry wt.) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . Total starch content in nodules (in mg/lS plants) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Concentration of starch in nodules (mg/g dry weight) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Total ethanol soluble carbohydrate content of primary roots (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . Concentration of ethanol-soluble carbohydrates of primary roots (mg/g dry weight) of sex sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . Total starch content of primary roots (in mg/15 plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . . . . . Concentration of starch of primary roots (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. . . . . . . . . . . . . . . . . . . . Total ethanol soluble carbohydrate content of "secondary roots" (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Concentration of ethanol-soluble carbohydrates of "secondary roots" (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars and corresponding Analysis of Variance . vi Page .108 .109 .110 .111 .112 .113 .114 O 115 .116 .117 .118 22. 26. 27. 28. 29. 30. 32. 33. 222. 23. 24. 25. 26. 27. 28. 29. 30. 32. 33. Total content of starch of "secondary roots" (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance . . . . Concentration of starch of "secondary roots" (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . Total content of ethanol soluble carbohydrates of stems (in mg/lS plants) of six sequential weekly harvests, of four dry bean cultivars, and corres- ponding Analysis of Variance . Concentration of ethanol soluble carbohydrates of stems (mg/15 plants) of six sequential weekly harvests of four dry bean cultivars, and corres— ponding Analysis of Variance . . . . . . . . . Total content of starch of stems (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Concentration of starch in stems (mg/g dry weight of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . Total content of ethanol soluble carbohydrates of leaves (in mg/lS plants of six sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance . . . . . . . . . . Concentration of ethanol soluble carbohydrates of leaves (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance . . . . . . . . . . Total content of starch of leaves (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . Total content of ethanol-soluble carbohydrates of pods (in mg/lS plants) of three sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . Concentration of ethanol soluble carbohydrates of pods (mg/g dry weight) of three sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . vii Page 119 120 121 122 123 124 125 126 127 128 129 36. ' 39. 43a. 42. I34. 35. 36. 37. 38. 39. 40. 40a. 41. 42. 42a. Total starch content of pods (in mg/lS plants) of three sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . . . . . Concentration of starch in pods (mg/g dry weight) of three sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . . . Total content of organic nitrogen in "primary roots" (in mg/plant) of ten sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance . . . . . . . . . . Concentration of organic nitrogen in "primary roots" (mg/g dry weight of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . Total organic nitrogen of "secondary roots" (in mg/plant) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . Concentration of organic nitrogen in "secondary roots" (mg/g dry weight of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . Total content of organic nitrogen of stems (in mg/plant) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . Cubic polynomials fitted to stem nitrogen content (dependent variable), and corresponding Analysis Of variance. 0 O O O O O O O O O O O O O O O I I 0 Concentration of organic nitrogen in stems (mg/g dry weight) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. . . . . . . . . . . . . . . . . . . . . Total content of organic nitrogen of leaves (in mg/plant) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . . . . . . . . . . . . . Cubic polynomials fitted to leaf nitrogen (dependent variable), and corresponding Analysis of Variance . . . . . . . . . . . . viii Page 130 131 132 133 '134 135 136 137 138 139 140 C ( 1n C . «IN; c l‘ s T I. I6. 58. In *1, 30. 51. 52. 53. 133. 44. 45. 46. 47. 48. 49. SO. 51. 52. 53. Concentration of organic nitrogen in leaves (mg/g dry weight) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . Total content of organic nitrogen of vegetative structures (in mg/plant) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance . . . . Total content of organic nitrogen (A) of flowers and rachis (in mg/plant) and concentration (B) (mg/g dry weight) of three sequential harvests of four dry bean cultivars . . . . . . . . . Total content of organic nitrogen (A) of pods (in mg/plant) and concentration (B) (mg/g dry weight) of three sequential harvests of four dry bean cultivars. . . . . . . . . . . . . . Analysis of variance of the effect of carbon dioxide on nitrogen fixation rates (u moles C2H4 5p1'l hr‘l) of the dry bean cultivar Porrillo Sintetico of two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). Analysis of variance of the effect of carbon dioxide on nodule fresh weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico of two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . Analysis of variance of the effect of carbon dioxide on nodule soluble carbohydrate concentration (mg./g. fresh weight) of the dry bean cultivar Porrillo Sin— tetico at 18 to 32 d.a.p. . . . . . . . . . . . . . . . Analysis of variance of the effect of carbon dioxide on nodule soluble carbohydrate (in gm/S plants) of the dry bean cultivar Porrillo Sintetico at 18 to 32 d.a.p. '0 O I O O O O O O O O O O O O O O O O O O O 0 Analysis of variances of the effect of carbon dioxide on root fresh weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico of two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B) . . . . . Analysis of variance of the effect of carbon dioxide on root soluble carbohydrate concentration (mg/g fresh weight) of the dry bean cultivar Porrillo Sin— tetico of two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . . . . . . . . . . . . . . Analysis of variance of the effect of carbon dioxide on root soluble carbohydrate (in mg/S plants) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . ix Page . 141 . 142 . 143 . 144 . 145 . 146 . 147 . 147 . 148 . 149' 150 3. L11 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. Analysis of variance of the effect of carbon dioxide on stem fresh weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico for two time periods: '18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). Analysis of variance of the effect of carbon dioxide on stem soluble carbohydrate concentration (mg/g fresh weight) of the dry bean cultivar Porrillo Sin- tetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . Analysis of variance of the effect of carbon dioxide on stem soluble carbohydrate (in mg/5 plants) of the dry bean cultivar Porrillo Sintetico for two time peirods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). Analysis of variance of the effect of carbon dioxide on leaf fresh weight (in grams/plant) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . Analysis of variance of the effect of carbon dioxide on fresh weight of pods (in g/5 plants) of the dry bean cultivar Porrillo Sintetico at 18 to 46 d.a.p.. Analysis of variance of the effect of carbon dioxide on root dry weight (in g/S plants) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B).. . . Analysis of variance of the effect of carbon dioxide an organic nitrogen concentration of roots (mg/g dry weight) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . . . . . . . . . . . . . . . . Analysis of variance of the effect of carbon dioxide an organic nitrogen content of roots (in mg/S plants) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . . . . . . . . . . . . . . . . Analysis of variance of the effect of carbon dioxide on stem dry weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B) . . . . Analysis of variance of the effect of carbon dioxide on organic nitrogen concentration of stems (in mg/g dry weight) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . . . . . . . . . . . . . . . . Page 151 153 154 ' 155 . 155 . 156 . 157 . 158 - 159 65. (3‘ ('7‘ c 68. 69. 64. 65. 66. 67. 68. 69. 70. Page Analysis of variance of the effect of carbon dioxide on organic nitrogen content of stems (in mg/g dry weight) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B) . . . . . . . . . . . . 160 Analysis of variance of the effect of carbon dioxide on leaf dry weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B) . . . . . . . 161 Analysis of variance of the effect of carbon dioxide on organic nitrogen concentration of leaves (in mg/g dry weight) of the dry bean cultivar Porrillo Sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B). . . . . . . . . . . . . . . . . . . . . . . 162 Analysis of variance of the effect of carbon dioxide on organic nitrogen content of leaves (in mg/S plants) of the dry bean cultivar Porrillo sintetico for two time periods: 18 to 32 d.a.p. (A), and 18 to 46 d.a.p. (B)............................163 Analysis of variance of the effect of carbon dioxide on pod dry weight (in grams/5 plants) of the dry bean cultivar Porrillo Sintetico at 18 to 46 d.a.p. . . '163 Analysis of variance of the effect of carbon dioxide on organic nitrogen concentration of pods (in mg/g dry weight) of the dry bean cultivar Porrillo Sintetico at 18 to 46 d.a.p. . . . . . . . . . . . . . . . . . . ' 164 Analysis of variance of the effect of carbon dioxide on organic nitrogen content of pods (in mg/S plants) of the dry bean cultivar Porrillo Sintetico at 18 to 46 d.a.p. O O O O O O O O O I O O O I I O O I O 164 xi M‘ '59. Ltd LIST OF FIGURES Page CHAPTER 2 l. Nitrogen fixation rates per plant of four dry bean cultivars taken at weekly intervals throughout ontogeny . . . . . . . . . . . . . . . . . . . . . . . . . 24 2. Nitrogen fixation (a) and ontogenetic distribution of dry weight (b): total (T); leaves (L); stems (S); flowers (F); pods (P); and nodules of the cultivar 72 VUL 26689 . . . . . . . . . . . . . . . . . . . . . . . 28 3. Nitrogen fixation (a) and ontogenetic distribution of dry weight (b) of vegetative (V) and, reproductive (RE) structures and, nodules (N) of the cultivar 72 VUL 26689 . . . . . . . . . . . . . . . . . . . . . . . 29 3a. Ontogenetic distribution of stem dry weight in the variety 72 VUL 26689. Values shown are derived from cubic polynomials. Inflection points (IF) and maxima (M) are shown. . . . . . . . . . . . . . . . . . . . . . . 30 4. Nitrogen fixation (a) and ontogenetic distribution of dry weight (b): total (T); leaves (L); stems (S); flowers (F); pods (P); and nodules (N) of the cultivar Porrillo Sintetico . . . . . . . . . . . . . . . . . . . . 31 4a. Ontogenetic distribution of leaf dry weight in the variety 72 VUL 26689. Values shown are derived from cubic polynomials. Inflection points (IF) and maxima (M) are shown. . . . . . . . . . . . . . . . . . . . . . . 32 5. Nitrogen fixation (a) and ontogenetic distribution of dry weight (b) of vegetative (V) and, reproductive (RE) structures and, noduels (N) of the cultivar Porrillo Sintetico. . . . . . . . . . . . . . . . . . . . . . . . . 33 6. Nitrogen fixation (a) and content of ethanol-soluble carbohydrates (broken line) and starch (solid line) (b) in leaves (L), stems (S), and nodules (N) of the cultivar 72 VUL 26689 . . . . . . . . . . . . . . . . . . . . . . . 38 xii 13. II. 16. 10. ll. 12. l3. 14. 15. 16. 17. Nitrogen fixation (a) and concentration of ethanol- soluble carbohydrates (broken line) (b) in leaves (L), stems (S), and nodules (N) of the cultivar 72 VUL 26689. Content of ethanol-soluble carbohydrates (broken lines) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar 72 VUL 26689 . Concentration of ethanol-soluble carbohydrates (broken line) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar 72 VUL 26689 . . . . . . . . . . . . . . . . . . . Nitrogen fixation (a) and content of ethanol-soluble carbohydrates (broken line) and starCh(solid line) (b) in leaves (L), stems (S), and nodules (N) of the cultivar Porrillo Sintetico . . . . . . . . . . . . . . . . . . . Nitrogen fixation (a) and concentration of ethanol- soluble carbohydrates (broken line) and starch (solid line) (b, c) in leaves (L), stems (S), and nodules (N) of the cultivar Porrillo Sintetico . . . . . . . . . . . . Content of ethanol-soluble carbohydrates (broken lines) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar Porrillo Sintetico. . . . . . . . . . . . . . . . . . . . . . . . Concentration of ethanol-soluble carbohydrates (broken line) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar Porrillo Sintetico. . . . . . . . . . . . . . . . . . . . . . . . Nitrogen fixation (a), organic nitrogen concentration (b) and content (c) of leaves (LN), stems (SN), primary roots (IAR), secondary roots (SAR), and pods (PN) of the cultivar 72 VUL 26689. . . . . . . . . . . . . . . . . Nitrogen fixation (a), organic nitrogen concentration (b) and content (c) of leaves (LN), stems (SN), primary roots (IAR), secondary roots (SAR), and pods (PN) of the cultivar Porrillo Sintetico. . . . . . . . . . . . . . Ontogenetic distribution of stem nitrogen. Values shown are derived from cubic polynomials. Inflection points (IF) and maxima (M) are shown. . . . . . . . . . . . . . . Ontogenetic distribution of leaf nitrogen. Values shown are derived from cubic polynomials. Inflection points (IF) and maxima (M) are shown. . . . . . . . . . . . . . . Page 38 4O 41 42 43 44 45 50 51 53 54 ”‘1 I. 4.. Al‘ 78 glib 2 I 26 18. 19. 20. 21. 22. 23. 24. 25. 26. Page Cross section of a Phaseolus vulgaris L. nodule at 53 dap with central bacterial cells (B), peripheral vascular bundle (V), cortex (C) and, uninfected cells (U) containing compound amiloplasts (A). Periodic Acid Schiff - stained section X40 . . . . . . . . 61 Cross section of a Phaseolus vulgaris L. nodule at 53 dap illustrating crenula (CR), vascular bundle (V), bacteriod containing cells (B), and uninfected cells (U) containing compound amyloplasts (A). Periodic Acid Schiff - stained section X400. . . . . . . . 61 Cross section of a Phaseolus vulgaris L. nodule at 53 dap with peripheral vascular bundles (V), bacteriod containing cells (B), uninfected cells (U) and, com- pound amyloplasts. Toluidine Blue-stained section. X100. . . . . . . . . . . . . . . . . . . . . . . . . . . 61 Cross section of a Phaseolus vulgaris L. nodule at 53 dap illustrationg bacteriod containing cells (B), nucleous (N), and uninfected (U) cells containing compound amyloplasts (A). Mercuric—Bromphenol Blue - stained section. 560x. . . . . . . . . . . . . . . 63 Cross section of a Phaseolus vngaris L. nodule vascular bundle at 53 dap with endodermis (E), casporian strips (CS), xylem (X), phloem (PH) and, compound amyloplasts (A). 800x. . . . . . . . . . . 63 Bacteriod containing cells (B) and uninfected cells (U) with nucleous (N), nucleolus (ns) and, compound amyloplasts (A). 850x. . . . . . . . . . . . . . 63 Cross section of stem at third node level illustra- tion amyloplasts (A) in secondary parenchyma cells. Phase-contrast photograph. 400x. . . . . . . . ... . . . 65 Cross section of stem at the third node level illustra- ting amyloplasts (A) in parenchyma cells of the pith. Phase-contrast photograph. 400x. . . . . . . . . . . . . 65 Cross section of stem at the third node level illustra- ting amyloplasts (A) primarilly located in the parenchyma cells of rays of secondary xylem. Periodic Acid Schiff - stained section. l40X. . . . . . . . . . . 65 xiv INTRODUCTION From the viewpoint of the agro-biologist, a bean plant can be defined as a biological system for the reduction and subsequent processing of nitrogen and carbon into forms useful not only to the plant in assuring its survival but to man as consumer of the bean seed. It is deemed important to man that the reduction and subsequent processing of nitrogen and carbon, as key elements in seed formation and seed quality, be conducted in the plant in as efficient a manner as possible. This requires understanding of the system. Great strides have been made in understanding soil nitrogen uptake, transport, and reduction, in symbiotic fixation of nitrogen, and in the synthesis and regulation of synthesis of nitrogen - containing compounds such as protein. Similarly, great strides have been made in understanding the photosynthetic process and in the transport and storage of carbon— containing compounds, for example, proteins, sugars, starch. In grain legumes, these processes are proceeding simultaneously and there is increasing evidence of interdependence among them. In particular, there is a hypothesis that the decline in nitrogen fixation in legume plants shortly after pod-filling commences is a result of competition by the developing seeds for photosynthate (energy) needed in the nodules to maintain a high rate of nitrogen fixation. There is also evidence that the form and/or ratio of carbohydrate forms stored in root nodules may influence the rate of fixation. Furthermore, different genotypes of legumes appear capable of differential fixation 'nO .35 m ”A I3 ..- I: rates, both of nitrogen and of carbon. The amount of starch storage in root and stem of bean genotypes appears to vary significantly among genotypes. These disclosures raise numerous questions and suggest several avenues of research, some of which form the bases of this thesis. It seems essential that the ontogenetic relationships that may exist between nitrogen fixation and carbohydrate partioning be documented in bean genotypes that differ in plant type and agronomic adaptation. The hypothesis of competition between nodule and developing pod should be looked at anew. The experiements conducted and reported in this thesis bear upon these problems. CHAPTER 1 SYMBIOTIC NITROGEN FIXATION I .In. 514:. a . “L: O .. " ‘hh Ext 33:? IL size It) INTRODUCTION Biological fixation of molecular nitrogen is considered a funda- mental phenomen in the maintenance of life. From the beginning of agriculture, symbiotic nitrogen fixation has been unwittingly utilized in the cultivation of legumes. In the latter part of the nineteenth century, the ability of legumes to grow on poor soils and to improve their fertility was finally explained after many contradictory and negative results. In 1886, Hellriegel and Wilfarth (cited by Virtanen and Miettinen, 1963) demonstrated experimentally that legumes utilize molecular nitrogen if their roots bear nodules induced and formed by bacteria. Two years later Beijerink (cited by Virtanen and Miettinen, 1963), isolated a bacterium in pure culture which formed nodules on the roots of the host plant. Our understanding of N2 fixation has advanced most rapidly during the past decade. This rapid advance in knowledge of the processes involved was due to major advances that include: a) recognition of ATP and electron requirements in E y_i_t_§2 assays (Hardy and D'Eustachio, 1964; McNary and Burris, 1962; and Mbrtenson, 1964); 6) separation of the protein components of nitrogenase (Bulen and LeComte, 1966), and, c) use of the acetylene-reduction assay, an indicator of nitrogenase activity (Dilworth, 1966; Schollhorn and Burris, 1966). To date, a concensus of opinion exists that essential biochemical requirements for N2 fixation are: a) nitrogenase; b) a source of reductant and electron carriers (reduced flavodoxin or ferredoxin and EC! 1 on» he. ‘ I 17»- -.l h u ‘HI . ‘ug‘; 4 NADPH); c) an energy source: ATP, derived from fermentation, substrate level phosphorylation, oxidative phosphorylation or photophosphory- lation, oxidative phosphorylation or photophosphorylation; d) a carbon backbone for amino acid and amide synthesis; e) a specialized pathway for assimilation of NH f) a regulatory system for nif genes; g) a 3; mechanism of oxygen protection for nitrogenase; and, h) hydrogen evolution. Among the above requirements three of them in particular, ATP, NADPH, and a carbon backbone for amino acids at the level of carbo- hydrate, have received considerable attention as possible limiting factors of the fixation process in legumes. ONTOGENETIC NITROGEN FIXATION Nitrogen fixation as a function of time has been found, for major cultivated legumes, to follow a typical sigmoid curve. A peculiar characteristic to all profiles of fixation reported relates to a losscmf activity during the ontogeny of legumes. The decline in some legumes coincides with the onset of flowering and pod-filling, whereas in other species (or different lines), the highest levels of N fixation appear 2 to occur during the seed-filling stage. Legume crops such as dry beans (Types I and II), (Day, 1972; Graham, 1975; and Dart gt_al,, 1976), peas (Munchin and Pate, 1973, LaRue and Kurz, 1973; Lawrie and Wheeler, 1973, 1974), and broad bean (Pate, 1958, Lawrie and Wheeler, 1974), have been reported to show a decline early in the pod-filling stage. Peanuts (Hardy ggflal., 1971) and cowpeas (Dart g; il°¢ 1976), in con- trast, show a rapid post-flowering increase in N fixation, continuing 2 throughout a considerable period of time into the pod-filling stage. IIndeterminate cultivars of dry beans (Graham, 1975), show a pattern of 8ustained maximum fixation rate during flowering and a large part of i“. .. Ob. . at? ‘Iv, '\5 s . v. u. uLi 5 the pod-filling stage. The presumed relationship between flowering and nitrogen fixation in soybeans appears to vary with the cultivar and location (Hardy EE'.§l°’ 1971; Mague and Burris, 1972; Lawn and Brun, 1974; and Thiobodeau and Jaworsky, 1975). It is conceivable that any one of the seven essential require- ments mentioned previously for nitrogen fixation to occur, could theoretically be considered as a potential limiting factor of this process. Several causes have been advanced as possibly limiting N2 fixation. These have been concisely summarized by Hardy and Havelka (1975). They propose that fixation may be limited by: a) concentra- tion of nitrogenase; b) percentage saturation of nitrogenase by sub- strates of the reaction, such as ATP, reductant or N and, c) uniden- 2 tified regulatory molecules. Inefficiency due to hydrogen evolution may also play an important role. The concensus of opinion in the literature, as shown below, favors the viewpoint that the amount of photosynthate available to the nodule may be the most significant factor limiting fixation and that a reduction in availability of photosynthate to these structures is a consequence of "intense competi- tion for photosynthate from reproductive structures". An important modification of this viewpoint appears to exist in dry beans and is expressed further in the context of this thesis. The hypothesis of carbohydrate supply as a primary factor in legume symbiosis has been entertained since the early 1930's. An example of the stage of knowledge at this time is the work of Allison (1930) and Allison and Ludwig (1939) which can be quoted as follows: "studies to date have shown that the available carbohydrate supply (chiefly sugars and starch) is a primary factor in determining nodule location, growth and size, quantity of nitrogen fixed by good strains, 6 disintegration of nodules, and similar phenomena" and, "the necessity for a supply of carbohydrate in legume symbiosis has long been recog- nized". Some years later, with the observations that inorganic nitro- gen usually has a negative effect on nodulation and the fixation prbcess, Wilson st} 21., (1940) attempted to combine into a single hypothesis (carbohydrate-nitrogen hypothesis) considerations of both the nitrogen and carbohydrate supply and their effects on legume growth. In the early studies no conclusive evidence was found that would relate carbohydrate supply to the decline in fixation rate, but they have undoubtedly served as the basis for current research. Evidence concerning this prevailing hypothesis can be analyzed primarily from two complementary points of view: first, evidence related to factors that increase photosynthetic output and; second, those which decrease the amount of available carbohydrate to the nodulated system. Consid— eration of the evidence demands that one should bear in mind that a subtle difference exists between factors that may be "nitrogen fixation limiting" pg£_§g and factor(s) that are direct or indirect causes of the observed decline in the activity of the system. A limited amount of information is available in the latter instance for the species under consideration in this thesis. As previously indicated, photosynthesis and carbohydrate levels in plants were recognized early on as important in symbiotic fixation of nitrogen. The demonstration of enhanced N2 fixation by CO2 fertilization in beans by Riedels in 1922 (cited by Allison, 1935), and in red clover by Wilson, E£-.2l°’ (1933) was neglected until the recent dramatic responses achieved with soybeans by Hardy and Havelka (1973, 1975). Riedels reported that the use of additional C02 increased the number of nodules on beans S-fold and increased plant growth. Wilson 7 st, gif, obtained increases of 100 to 200 percent in the dry weights and in nitrogen fixation when the pCO was increased from that in 2 normal air to 0.2 or 0.4 percent. The plants had two or three times as many nodules as the controls in air, and their size was greatly increased. Similarly, nitrogen fixation and nitrate reductase activities were measured by Hardy and Havelka (1973) at weekly inter— vals on field grown soybeans. Plants were exposed to air and C02- enriched air (800-1200 ppm C02) during the day, from preflowering to maturity. Carbon dioxide enrichment according to these authors resulted in a) doubled specific activity of nodules, b) doubled nodule fresh weight and, c) a longer exponential phase and delayed loss of activity. In contrast, nitrate-reductase activity decreased by 65%. Nitrogen fixation accounted for 85% of the nitrogen in mature plants under C0 -enrichment but only 26% in air. No data were provided 2 in this short communication in 1973. In 1975, data provided relates to various parameters at maturity and supported their conclusions only in part for: a) increased specific nodule activity (.228 mg N/g nodule dry weight to 1.187 mg N/g nodule dry weight); and, b) a longer exponential phase of fixation. Nodule mass reported was not doubled. Evaluation of the evidence is hampered since two different plant populations were used for air and C0 treated plants (455,000 plants/ 2 ha. for air-controls and 505,000 plants/ha. for CO2 - treated plants). They suggested that the elevated pCO2 increased net photosynthesis, due mainly to a decrease in photorespiration. Quebedeau, 23‘ al., (1975) reported similar results concerning pCO2 effects on nitrogen fixation and dry matter production. They found that N2 fixed was increased by C02/02 ratios greater than those 8 of air and was decreased by ratios samaller than those of air. Treat— ‘ment combinations of 300 ul C02/1 with 5 and 10% 02 resulted in rela- tive increases to air of 125% and 50% respectively. A treatment com- bination of 1200 ul C02/1 and 21% 0 yielded a 339% increase over the 2 air-control soybean plants. They proposed that such an increase in total nitrogen fixed and total growth by subambient 02 and C02-enrich- ment can be attributed to a reduction of photorespiration, and a direct 02 inhibition of photosynthesis. Data reported at maturity shows that nodules were still present at that time. Other treatments such as low planting density, increasing source size by grafting, supplemental light and pod removal have, in general, been found to increase N2 fixation rates and other associated para- meters. Streeter (1974) reported increases of 75% over grafted controls in nitrogen fixation due to doubling the shoot:root ratio, while dry weight of nodules increased by 30% over control plants. In contrast, Lawn and Brun (1974) reported no increase in nodule fresh weight due to grafting or in the specific activity of nodules. The reported values for total activities of root genotypes appear to be significant. Whereas, Streeter utilized soybean plants grown in a sand-culture, nitrogen-free medium, Lawn and Brun utilized field-grown soybean plant-s- Lawn and Brun (1974) reported a positive response of nodule activity and protein yield for two soybean varieties to supplemental light during the day. Partial depodding (removal of all pods from alternate nodes), resulted in 3.3% and 1.9% increases in seed protein and 18.9% and 12.1% in nodule activity over control plants (Chippewa 64 and Clay varieties, respectively). They suggested that the responses 9 indicated previously are consistent with the hypothesis of a limita- tion to symbiotic nitrogen fixation by competition for photosynthates from developing pods. Bach gt, aI., (1958) before the advent of the acetylene-reduction assay, reported that specific radioactivity in nodules as compared to roots (after exposure to "several hours" to 14C02), was higher by a factor of 1.34 and 2.08 for day and night samples. Exposure of excised and/or crushed nodules to 15NZ to determine the possibility of restoring their activity upon the addition of sucrose, fructose and glucose, resulted, according to them, in increases in fixation. Their data supports their conclusion only for the addition of sucrose in one of the experiments reported. Two additional experiments, one in which the three sugars were tested, and another in which sucrose alone was tested, do not support their contentions. Wong and Evans (1971) also working with soybean plants, reported that the addition of sucrose, fructose, glucose, pyruvate, succinate or malate to nodules exhibiting low nitrogenase activity failed to restore their activity. Mbre recent literature in whichll‘CO2 labelling has been used provides, in some instances, more direct evidence for the hypothesis under consideration. In 1974, Lawrie and Wheeler, working with pea plants, reported data on experiments concerning the extent to which "photosynthesis of the plant can satisfy the competition for assimilates between different metabolic sinks". They studied changes in nitrogenase activity and accumulation of photosynthates in the nodules during flowering and fruit formation. They reported that maximum nitrogenase activity and specific activity of nodules occurred 3 weeks after planting at flowering, and subsequently declined during fruit development. ll . 2VII. and. EXPO Vity and tie: III lO Senescence of nodules was evident from the presence of green nodules in the fourth week. Total radioactivity of plants doubled between weeks 3 and 4 to reach a maximum in week 4. Radioactivity levels in nodules, roots, leaves (1 through 7), and flower apices was determined for 2, 3 and 4-week old plants. Stems were not analyzed. In their discussion section they claim to have obtained a 60% decrease in specific activity of the nodules as the plants entered the reproductive phase, following a 6-hour cold-chase period with carbon dioxide. But, in the figures depicting the distribution of CIA-labelled photosynthates in relation to plant age, no differences in the radioactivity of the nodules after 6 and 25 hour cold-chase periods in any one of the 2, 3, and 4 week-old plants can be detected. The alternative approach in the literature to evaluating the hypothesis under consideration has been to assess the effect of those treatments that reduce the supply of "energy" to the nodulated system. Those that seem to be more relevant are the effect of removal of "competitive sinks", reduction of source size and low light effects. Hardy £5..§I., (1968) reported on the effect of leaf removal and exposure to toal darkness of soybean plants. Acetylene-reducing acti- vity decreased 12% relative to the control after 1 day and was 14% of‘ the control plants even ten days after leaf removal. Exposure to 17 hours of darkness resulted in a 70% decline in nitrogenase activity with respect to control plants. Similar results of the effect of leaf removal were reported by Lawn and Brun (1974) for soybeans. Lawrie and Wheeler (1973) working with Piggy reported that a marked correla- tion was found between the accumulation of photosynthates in nodules and the rates of acetylene reduction during the growth cycle of pea 4 5“. . III: ...4 q . 4.. a u v 0.. 9L a a}. A... “A. u s h. .5 IV .3 ~ . e a a. nod ’v-‘as C011 it] A 11 plants. When plants were maintained in the dark for periods of 38, 62, and 72 hours and subsequently returned to the light, nitrogenase activity recovered in plants exposed to 38 hours darkness almost to the level of plants maintained in continuous light. Plants darkened for periods of 62 and 72 hours did not fully recover. Radioactivity levels in nodules were similar to those of plants maintained in continuous light for the 38 hour dark-treated plants, but not for the 62 and 72 hour dark-treated plants. It is unfortunate that an inappropriate statistical treatment of the data does not allow one to determine the validity of the results reported. Also, it would appear that the appropriate control to use for comparison is not plants subjected to continuous light but plants grown in a normal light regime. In another experiment conducted on the effect of darkening on the rate of acetylene reduction, nitrogenase activity was found to decline, upon exposure to darkness, to near zero, while radioactivity level in the nodule appeared to decrease only after 72 hours exposure to darkness. But, the radioactivity level in control plants kept in continuous light was lower than that of darkened plants! They proposed that the continued increase of radioactivity in the nodules of darkened plants up to 48 hours suggests that "a substantial proportion of nodule photo- synthate must also be used to support other activities such as growth and multiplication of the endophyte". It is interesting that no positive correlation between radioactivity level and the loss of acetylene reduction activity was found until 72 hours darkness which also could be interpreted as an indication that photosynthate pgr_§g_is not "limiting". The most direct kind of evidence found to support the idea that nitrogen fixation might be reduced as a consequence of reduced energy 12 supply to the nodules has been recently published by Ching, et. al., (1975). Working with detatched soybean nodules, they reported that exposure of soybean plants to a l-day dark treatment resulted in reductions of 50% in nitrogenase activity in the nodule tissue, 15% of the energy charge, 60% of the sucrose content, 70% of the ATP con— tent, 60% of total adenosine phosphate content and 55% of the ratio of ATP/ADP. Longer periods of darkness resulted in further decreases. The effect of removal of "competing sinks" on N2 fixation has received considerable attention in recent literature; the results reported showing discrepancies but in general supporting the view that removal of pods and/or flowers usually results in maintenance of reducing activity of nodules. Hardy E£!.El°’ (1968) reported no positive effect due to pod removal, in fact, they report decreases in nitrogenase activity and nodule weight due to pod removal. In con- trast, Lawrie and Wheeler (1974), found in peas that the continuous removal of flowers "prevented decrease in acetylene-reduction activity". Nodule weight of intact plants was shown to be three times larger in 5-week old plants. Lawn and Brun (1974) found similar positive effects on N2 fixation due to partial pod removal in soybeans. They also reported that a stage is reached in soybean plants when the growth rate of pods equals that of the tops and at this stage all of the dry matter increase in the tops is accounted for by the increase in pod dry matter (Note: vegetative organ competition is not mentioned). Subsequent to this stage, pod growth was found to exceed the growth rate of tops, indicating mobilization and translocation of previously stored assimilates from other plant parts into pods. Decline of nodule activity occurred immediately prior to the stage at which pod growth equalled the growth rate of the tops. They also proposed that this 13 can be taken as evidence that reduction of symbiotic activity is associated with development of the pod as a strong assimilate sink. Their suggestion is compatible with the observation of Hume and Crisswell (1972) that only small amounts of label were recovered from the roots and nodules after pod filling commences. Similarly, with peas, Minchin and Pate (1973) reported that nOdule senescence coincides with the initiation of flower primordia. They speculated that it is possible that nodules "fare badly in competition with the root for assimilates" during the reproductive period. With dry beans, Dart (1974) reported that nodules of many dry bean varieties senesce at flowering (in contrast to soybeans and cowpeas) with a new population of nodules forming during the early pod filling period. Knowledge concerning the possible causes of nodule senescence is limited. No known studies have revealed the internal cause(s) respon- sible for this phenomenon. Klucas (1974) found no significant changes in dry weight or total nitrogen in tap root nodules of soybeans during the period in which permanent loss of nitrogenase activity was observed. Total leghemoglobin content did not decrease during the initial decline in N2 fixation. In contrast, poly-B-hydroxybutyrate was found to accumulate as the nodules aged, a significant increase being reported prior to or concomitant with the decrease in nitrogenase activity. The significance of this polymer remains unknown. It has been proposed that such a polymer could be an energy source, but Wong and Evans (1971) have shown that the presence of a supply of poly-B-hydroxbutyrate in soybean nodule bacteriods is not sufficient for maintenance of high nitrogenase activity under conditions of limited carbohydrate supply from the host plant, i.e.,a) incubation of plants in the dark (up to 16 days); b) incubation of excissed nodules in dark (up to 60 hours); and c) the onset of senescence of nodules. 10. ll. 14 REFERENCES Allison, E. F. and C. A. Ludwig. 1939. Legume nodule development in relation to available energy supply. J. Amer. Soc. Agron. 31: 149-158. Allison, F. 1935. Carbohydrate supply as a primary factor in legume symbiosis. Soil Sci. 31: 123—143. Bach, M. K., W. E. Magee and R. H. Burris. 1958. Translocation of photosynthetic products to soybean nodules and their role in nitrogen fixation. Plant Physiol. 118-124. Bulen, W. A. and J. R. LeComte. 1966. The nitrogenase system from Azotobacter: Two enzyme requirements for N reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc. Nat. Acad. Sci. USA. 56: 979-986. Ching, T. C., S. Hedtke, S. A. Russell and H. J. Evans. 1975. Energy state and dinitrogen fixation: soybean nodules of dark-grown plants. Plant Physiol. 55: 796-798. Dart, P. J., J. M. Day, A. R. J. Eaglesham, P. A. Huxley, F. Minchin and R. J. Summerfield. 1976. In "Joint FAO/IAEA Coordinated Programme on the Use of Radioiso- topes in Fertilier Efficiency Studies in Grain Legumes." Vienna, Paris. Day, J. M. 1972. Studies on the role of light in legume symbiosis. Ph.D. thesis. University of London, England. Dilworth, M. J. 1966. Acetylene reduction by nitrogen-fixing preparations from clostridium pasterianum. Biochim. Byophs. Acta. 127: 285-294. Graham, P. H. 1975. In "Annual Report". Centro Internacional de Agricultura Tropical, CIAT, Cali, Colombia. pp. C24-C29. Hardy, R. W. F. and U. D. Havelka. 1975. Photosynthate as a major factor limiting N2 fixation by field grown legumes with emphasis on soybeans. In "Symbiotic Nitrogen Fixation in Plants." P. S. Nutman, ed. Cambridge Univ. Press. New York. pp. 421-439. Hardy, R. W. F. and U. D. Havelka. 1973. Symbiotic nitrogen fixation: multifold enhancement by C02-enrichment of field grown soybeans. Plant Physiol. 51: 535. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 15 Hardy, R. W. F., R. C. Burus, R. R. Hebert, R. D. Holsten and E. K. Jackson. 1971. Biological nitrogen fixation: A key to world protein in "Biological nitrogen fixation in natural and agricultural habitats". Lie, T.A. and E. G. Mulder, eds. Plant and Soil: 561-590. Hardy, R. W. F., R. D. Holsten, E. K. Jackson and R. C. Burus. 1968. The acetylene-ethylene assay for N2 fixation: labora- tory and field evaluation. Plant Physiol. 43: 1185-1207. Hardy, R. W. F. and A. J. D'Eustachio. 1964. The dual role of pyruvate and the energy requirement in nitrogen fixation. Biochem. Biophys. Res. Commun. 15: 314-318. Humme, D. J. and J. G. Crisswell. 1972. Translocation and res- piration losses in soybean after assimilation of 14C02 at various growth stages. Agron. Abst. 35. LaRue, T. A. G. and W. G. W. Kurz. 1973. Estimation of nitro- genase in intact legumes. Canad. J. Microbiol. 19: 304-305. Lawn, R. J. and W. G. W. Kurz. 1974. Symbiotic Nitrogen fixa- tion in soybeans. I. Effect of photosynthetic source-sink manipulations. Crop Sci. 14 : ll-l6. Lawn, R. J. and W. A. Brun. 1974. Symbiotic nitrogen fixation in soybeans. III. Effect of supplemental nitrogen and inter- varietal grafting, Crop Sci. 14: 22-25. Lawrie, A. C. and C. T. Wheeler. 1973. The supply of photo- synthetic assimilates to nodules of Pisum sativim L. in relation to the fixation of nitrogen. New Phytol. 12: 1341-1348. Lawrie, A. C. and C. T. Wheeler. 1974. The effects of flowering and fruit formation on the supply of photosynthetic assimilates to the nodules of Pisum sativum L. in relation to the fixation of nitrogen. New Phytol. 73: 1119-1127. Lawrie, A. C. and C. T. Wheeler. 1974. Nitrogen fixation in the nodules of Vicia faba L. in relation to the assimilation of carbon. New Phytol. 74: 429-436. Mague, T. H. and R. H. Burris. 1972. Reduction of acetylene and nitrogen by field grown soybeans. New Phytol. 71: 275-286. McNary, J. E. and R. H. Burris. 1962. Energy requirements for nitrogen fixation by cell-free preparations of clostridium pasterianum. J. Bacteriol. 84 : 598-599. Miuchin, F. R. and J. S. Pate. 1973. The carbon balance of a legume and the functional economy of its root nodules. Journ. Expnt. Bot. 24: 259-271. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 16 Mortenson, L. E. 1964. Ferredoxin and ATP, requirements for nitrogen fixation in cell—free extracts of Clostridium pasterianum. Proc. Nat. Acad. Sci. USA. 52: 272-279. Pate, J. S. 1958. Nodulation studies in legumes. I. The synchronization of host and symbiotic development in the field pea, Pisum arvense L. Aust. J. Biol. Sci. 366-381. Quebedeaux, B. and R. W. F. Hardy. 1975. Reproductive growth and dry matter production of Glycine max (L) Merr. in response to oxygen concentration. Plant Physiol. 55: 102-107. Quebedeaux, B., U. D. Havelka, K. L. Livak and R. W. F. Hardy. 1975. Effect of altered p02 in the aerial part of soy- bean on symbiotic N2 fixation. Plant Physiol. 56: 761-764. Riedels, F. 1922. Kohlensauredungung. Technik. Landw. 3: 87-89. Schollhorn, R. and R. H. Burris. 1966. Study of intermediates in nitrogen fixation. Fed. Proc. 25: 710. Streeter, J. G. 1974. Growth of two soybean shoots on a single root: effect on nitrogen and dry matter accumulation by shoots and on the rate of nitrogen fixation by nodulated roots. J. Exp. Bot. 25: 189-198. Thiobodeau, P.S. and E. G. Jaworski. 1975. Patterns of nitrogen utilization in the soybean. Planta. 127: 133-147. Wilson, P. W., E. B. Fred and M. R. Salmon. 1933. Relation between carbon dioxide and elemental nitrogen assimilation in leguminous plant. Soil Sci. 35: 145-165. WOng, P. P. and H. J. Evans. 1971. Poly-B-hydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity. Plant Physiol. 47: 750-755. Virtanen, A. I. and J. K. Miettinen, 1963. Biological nitrogen fixation. In "Encyclopedia of Plant Physiology." F. C. Steward, ed. 5: 539-668. CHAPTER 2 CARBOHYDRATE PARTITIONING AND NITROGEN FIXATION PHASEOLUS VULGARIS L. Abstract The ontogenetic relationships that may exist between symbiotic nitrogen fixation and carbohydrate partitioning were studied in four dry bean cultivars. Total soluble carbohydrate and starch contents of the primary nodule population were found to be closely correlated in time with nitrogen fixation activity. The decline in nitrogen fixation activity was found to be correlated with loss of nodule dry weight, decreased total soluble carbohydrate and starch of nodules and an increase in soluble carbohydrate and starch in leaves and stems during the early stages of reproductive growth. Starch concentra- tion was found to decline one week prior to the occurrence of nitrogen fixation maximuma suggesting that hydrolysis of this polysaccharide may contribute energy to the fixation of molecular nitrogen. Reduction of carbohydrate movement to the nodule, due to competition by repro- ductive sinks, may not be the cause of the observed decline in nitrogen fixation. Temporary sites of starch accumulation were found to be restricted primarily to parenchyma cells of secondary xylem and pith in stems and to uninfected cells in nodules. l7 18 INTRODUCTION The ontogenetic decline in the rate of nitrogen fixation in many legumes has been clearly documented (2, 3, 4, 6, 7, 8, 9, 10, ll, 12, 14, 15, 20, 21, 22, 23). A decreased supply of photosynthate to the nodules has been advanced as a possible cause for the drop in fixation during the initial phases of the reproductive period (6, 8, 10, 11, 12, 13, 22, 23). The coincidence of decreased nitrogenase activity with a given stage of development in the legume under consideration has prompted several investigators to deduce that "competition" for photo- synthetic assimilates by the developing reproductive structures has a direct negative influence on the energy supply to the nodules. The purpose of this study was to evaluate the hypothesis based on reported research that a change in the pattern of partitioning of carbohydrates between vegetative and reproductive sinks is correlated with or is the ultimate cause of an observed decline in the rate of nitrogen fixation in Phaseolus vulgaris L. MATERIALS AND METHODS Plants utilized in this study were grown at the experimental site "Las Guacas" of the Secretaria de Agricultura del Valle del Cauca, located in Popayan, Colombia. This site has been used by the Inter— national Center for Tropical Agriculture (CIAT) in Cali, Colombia, as its major location for nitrogen fixation studies in dry beans. The experimental areas were fertilized on September 16, 1975 by broadcasting the following fertilizer equivalents per hectare: 1,000 kg of triple superphosphate; 50 kg KCL; 0.5 kg NaMoOA; 7.0 kg ZnSOa; 2.0 kg Borax; 1,000 kg of agricultural lime; 100 kg MgSOa; and 2.0 kg CuSO4. The experimental area consisted of a section 30 by 70 m. The I “I: ‘V.- ts I A: V \U. an '43 at II II l9 fertilizer was incorporated by disc-harrowing. A split-plot experi- mental design with randomized blocks was utilized,harvests CU1harvests) were the main plots and cultivars (4 cultivars) were sub-plots. Four replications were used. The experimental unit consisted of 4 rows. 4 m long per harvest per cultivar. Planting distances were 50 cm between rows and 7.5 cm between plants in the row. Four cultivars were selected for this study according to their previously determined nitrogen fixation capability. The cultivars selected were "72 VUL 26689" , "ICA Pijao", "NEP-Z", and "Porrillo SinteticoN. All four cultivars are of semideterminate (Type II) growth habit. Seed of the four cultivars was inoculated with an efficient Rhizobium phaseoli strain, CIAT 57, and pelleted immediately prior to hand planting. The ”peat" (turba)-based inoculum was bouned to the seeds with a 40% Cum Arabic aqueous solution and pelleted with tech~ nical grade CaCO Seeds were sown on September 19. 1975. 3. Acetylene - Ethylene reduction assays and sample preparation. Plants of each variety were harvested at weekly intervals beginning 25 days after planting. Assays were conducted between 10 and 12 a.m. Fifteen plants per variety per replicate were carefully uprooted. Five nodulated roots were used per variety for each acetylene assay. The tops of these five plants were placed in paper bags and stored in styrofoam coolers containing dry ice until further processing of the samples. This subsample of five plants was utilized for chemical determinations after separation into component parts and drying. Drying was accomplished in 2 days at 60 C. Subsequently, plant parts were weighed and ground in a Wiley Mill to pass a 60—mesh screen. Ground samples were stored in 25 by 125 mm teflon-lined screw :0 e1 .r. \ Ann ~ I ..h A: s is“ 20 cap culture tubes until analyzed. The remaining ten plants of each sample were similarly treated (with the exception of grinding) for complete growth analysis. Combining weights of the 5 plants used in the acetylene assays with weights of the 10 plants indicated above allowed expressing values in grams/15 plants. Assays were performed in dark-brown, 1 1 glass jars having a vacutainer stopper in the center of the lid and a gas tight seal. One tenth of an atmosphere of acetylene (welding grade) was injected after removing an equivalent amount of air. Incubation time was 30 minutes. A 10-ml gas subsample was transferred into vacutainer tubes at the end of the incubation period. Ethylene analysis was performed with a Perkin—Elmer Gas Chromatograph with 3 Flame Ionization Detector. A 1.8 m. stainless steel column packed with Porapak N was used. Calibra— tion of detector response was obtained by using a 98 ppm C2H4 standard. Volume of sample injected was 1 ml. Analysis gf_total organic nitrogen. One hundred milligram subsamples of dried and ground plant tissues were analyzed by micro-kjeldhal following the procedure of Yoshida, gt. al., (1971). Analysis gf_sugars (ethanol-soluble carbohydrates) and starch. Ethanol-soluble and insoluble (starch) carbohydrates were deter- mined quantitatively by a modification of the antrhone methodcfifYoshida EE‘.§£°’ (1971). The modified procedure is as follows: a subsample Of approximately 100 mg dried tissue was extracted three times for 30 minutes each with 80% analytical grade ethanol in a constant tempera— ture water bath (80 C). After which each sample was centrifuged (2000 X g). Tubes were capped with glass balls during extraction. Ethanol extracts were combined and adjusted to 30 m1 volumes. The 21 residue remaining was used for starch determinations as indicated below. An aliquot (usually 1 ml) of extract was diluted (20X to 50X) with deionized-distilled water. Two ml aliquots of this diluted extract were transferred into 25 by 150 mm pyrex screw capped culture tubes and the tubes were placed in an ice-water bath for 10 minutes. Four ml of anthrone reagent (0.5 g of anthrone in 1000 ml of analytical grade sulfuric acid, 95%, aged in the dark for 1 hour prior to use) were added by running the reagent on the side of the test tubes and kept in ice-water bath for five more minutes. Subsequently, test tubes were swirled with a test tube agitator and chromogen development was performed in a water bath (100 C), for exactly 7 minutes. Absorvance was determined using a Spectronic-ZO colorimeter, using a blank containing deionized—distilled water plus the antrhone reagent at 630 nm. A set of five standards (0.0, 0.025, 0.050, 0.075 and 0.100 mg glucose/2 ml) were run every time a set of samples was analyzed. The residue remaining after ethanol extraction and centrifugation was dried overnight at 80 C. Following this, 2 m1 of deionized-dis- tilled water were added to each sample, stirred in a test tube agitator and placed in a water bath at 100 C for 15 minutes. Samples were then allowed to cool to room temperature and 2 ml of 9.2 N HCLO4 were added and stirred occassionally for 15 minutes. Volume was adjusted to 10 ml with deionized-distilled water and centrifuged at 2000 x g. A second extraction was performed with 2 m1 of 4.6 N HCLO . adjusted to 10 ml 4 volume and centrifuged. The combined extracts were adjusted to 20 ml volume and chromogen development with anthrone was performed as indicated previously. Chemical analysis of samples strictly adhered to the experimental design utilized in the field. 22 Histochemical Analysis 9f Nodules and Stems. The procedures concerning the fixation of nodule and stem samples, the dehydration, embedding in plastic, and staining with Toluidine Blue and Periodic Acid Schiff Reagent have been carried out after Feder and O'Brien (1968). According to O'Brien and McCully (1969), Toluidine Blue “binds with carboxylated polysaccharides (pectic acids) to give a pinkish purple color and to molecules with free phosphate groups (for example, nucleic acids), give purplish or blue green. Hydroxylated poly— saccharides, such as cellulose and starch are not stained by this dye. DNA stains blue or blue green and RNA purple. The mercuric—Bromphenol Blue procedure was carried out after Mazia gt. El" (1953), with some modifications as follows: 1) 0.1% Bromphenol Blue in saturated aqueous HgCl - 20 minutes. 2 2) 0.5% acetic acid in saturated aqueous HgClq - 10 minutes. I. 3) Sections immersed (not rinsed) in demineralyzed water (pH 7.0), two changes, one minute each. M3213.E£°.§l" (1953) reported that either an aqueous or alcoholic solution of Bromphenol Blue in saturated HgCl can be used in the above 2 procedure. The aqueous solution was selected since an alcoholic solu— solution caused plastic infiltrated sections to slide-off the glass slides. According to Ruthman (1970), proteins are stained intensely blue. RESULTS AND DISCUSSION Symbiotic nitrogen fixation during ontggeny Analysis of the results obtained for the four dry bean cultivars 72 VUL 26689 (C1), ICA Pijao (C2), NEP-2(C3) and Porrillo Sintetico (C4) 23 revealed a close similarity in trends. These consistent patterns exhibited for almost all parameters measured has lead to restricting the presentation of results and disucssion to two of the four cultivars studied. The cultivars to be considered in detail are 72 VUL 26689 and Porrillo Sintetico. The results obtained for the four cultivars and their appropriate statistical analyses are presented in the Appendix Tables. Significant departures from the observed general trends are indicated when present. Seasonal profiles of nitrogen fixation activity are shown in Figure 1. Corresponding values and Analysis of Variance are shown in Appendix Table 10. A peculiar characteristic of the profiles is the sharp decline in nitrogenase activity of the cultivars ICA Pijao (C2) and NEP-2 (C3) at 67 days after planting henceforth referred to as (dap), i.e., at harvest 7. In contrast, the cultivars Porrillo Sintetico (C4) and 72 VUL 26689 (C1) exhibit this decline seven days later, at 74 (dap) (harvest 8). Macroscopic evidence of the onset of the reproductive period (as indicated by the presence of open flowers) in all four cultivars was present at 53 dap (harvest 5). One can deduce that the decline in fixation occurs during the initial phases of pod development. The cultivar NEP-2 had a growth cycle of 112 days, whereas the other three cultivars had a growth cycle of 116 days. The cultivars 72 VUL 26689 and Porrillo Sintetico sustain maximum fixation rates for at least a 7-day period. In contrast, NEP-Z and ICA Pijao reached a maximum fixation level at 60 dap (harvest 6) and subsequently declined to approximately the same level present at 39 dap (harvest 3). A small burst in nitrogen fixation activity for the four cultivars was observed at 81 dap (harvest 9). Similar activities, due possibly to "secondary nodule population levels", have 24 .zcowOuco uaonwsounu mam>umucw zaxwms um cmxmu mum>fiuflso came mun ~50w mo ocean non mouse cofiumxww cowouuwz .H ouswwm mcfiucmaa nouwm when mm Hm «A no so mm we mm mm mm b a p P _ p L N b \ on ‘0‘. ‘ \ 3 all. lie-II. .MV . ./ \.\ an // . .\. turn/ll \u /o/ .\.\ l!?$‘9+§0§9dl\ I. O ‘ \ ul/ / \\ “A“ o \o‘. nil \\ ..u//\\\aau /. \. In \ why“..- I /o o It |\|\.\ \ o p C , , \ ...u\ .3 I . . aka \ I /o \ $\\ . i ’ x \ .\ . a /’ /. \ ‘\ I /. \ six ) I /o \ ‘$\ A z . ax H / o \ ‘\ r I /n \o “\ 1| 40 I /. o $ \ ON e z < as s a z a a so / N 4 x s .. , .. . 3+:.¢+/ a coaumucam OHkuuom .+o¢++ I“. on \ N I mmz |.I.l. . a 03.: <3 III I H <1 .S> «a Ion (I_1q I {d vuzg satom n) uoInexI; uaBOJJIN 25 been previously reported by Dart (1974) and Graham (1975), for dry bean cultivars. Estimates of milligrams of N fixed/plant were 2 computed using the following relationship (see Table l): mg N2 fixed = 1(umoles C2H4 X 24 hr X 7 days — 1 t plwk 3 plhr 1 wk ) ”pans where 0.333 = theoretical conversion factor of mg C2H4 produced/mg N2 fixed. 0.81 = diurnal factor for nitrogenase activity. They indicate that 72 VUL 26689 and Porrillo Sintetico fixed 30.67% and 26.86% of the total symbiotically-fixed N prior to the appearance 2 of open flowers. Similarly, 69.33% was fixed by 72 VUL 26689 and 73.13% by Porrillo Sintetico from 53 dap onwards, i.e., in the reproductive phase of development. Values for ICA Pijao and NEP-Z were 24.86% and 23.17% prior to macroscopic flowering, and 75.13% and 79.82% during the reproductive phase. The highest fixation levels per week occurred at harvest 6 and 7 for the four varieties. During these two weeks, 72 VUL 26689 fixed 50.56% (73.28 mg N2), ICA Pijao fixed 48.75% (63.63 mg N2), NEP-2 fixed 49.91% (39.84 mg N2) and Porrillo Sintetico fixed 47.62% (67.58 mg N2) of the total N2 fixed during their ontogenies. Table 1 shows that 72 VUL 26689 and Porrillo Sintetico were the highest fixers. Better fixation in these cultivars came about pri- marily from maintaining their fixation activity at their maximum levels for 2 weeks. The cultivar 72 VUL 26689, the best fixer, also shows higher rates of fixation than the other three cultivars prior to the period in which N2 fixation rates reach maximum levels. Fixation levels beyond the point of decline in maximum activity can be observed 26 .wcwcmHm “some how "a oo.ooH Nm.HsH co.ooH mm.a~a oo.ooa mm.¢ma oo.ooa ea.esa Hence o~.m mm.s mH.s mm.m mm.~ an.m e~.o am.o we om.a so.HH mm.~H so.oa we.“ mm.m mm.o RN.H Hm N~.s ss.m sm.m mm.~ Hm.s m~.e km.m am.s as os.m~ H~.mm os.sH mN.NH em.ma sm.s~ ee.m~ aa.am so ~N.¢~ am.sm Hm.nm He.am Hm.m~ so.mm os.e~ mo.om oe m~.oH oe.s~ Hm.m mm.» mm.HH Hn.mH e~.sH ae.o~ mm He.ma em.HN ma.ma RH.HH oo.a Ne.HH me.oa ss.ma es Ne.m as.m mm.m Hm.m mm.a m~.oa ma.» Hw.HH an m~.s no.0 so.m mm.~ nm.a ~o.~ mm.n so.» NM on.m mo.s mo.m ~o.~ os.o on.» o~.e «H.s nN Aeneas no no Ass\eav Asuuou no as Ass\mav Assoc» no as Aas\mav Aauuou no No Aaa\mav cause «2 cause Nz amuse Nz amuse Nz Haeaev ousuoueam oHHauuoa ~Iauz omega <6H mmoe~ 49> «a was» uuu>uum mum>wuaao .mum>fiuH:u anon hum know we mHm>uounH maxoos Hmausoauon sou mo noxam somehow: Howey mo owmuaouuoa can noxwm cowouuwaan .H nanny 27 to remain low, but appear not to reach a zero level, probably indica- ting that nodules induced in more advanced stages of development of the bean cultivars, could account for this sustained but low activity. Partitioning_gf.d£y matte: Figures 2, 3, 4, and 5, show that total plant dry weight increased continuously up to 81 dap (harvest 9) in the cultivars 72 VUL 26689 and Porrillo Sintetico. Nodule dry weight increases occur up to 60 dap (harvest 6) and subsequently decline at 67 dap (harvest 7) and 74 dap (harvest 8) for 72 VUL 26689 and Porrillo Sintetico, res- pectively. At harvest 9 (81 dap), a small increase in nodule dry weight was again observed in all four cultivars. This increase in nodule mass is consistent with the observed increase in fixation rates observed at harvest 9. Maximum fixation rates can be seen to coincide with maximum dry weights of the nodule population, one week after the appearance of open flowers (53 dap). These trends were exhibited by all four varieties as shown in Appendix Tables 1 through 9 and 11. Dry weights of leaves increased up to 67 dap (harvest 7). A small decrease in dry weight of leaves was observed at 74 dap (harvest 8) in all cultivars. This decrease in dry weight of leaves appears to be due primarily to leaf fall, a characteristic of dry bean varieties of determinate and semideterminate growth habit. Leaf senescence and abscission appears to be restricted primarily to the lower leaves of been cultivars. Data from component parts revealed that at the cotyledonary scar positions, new branches and leaves are formed from 53 dap on. This fact may be important in the carbohy- drate distribution pattern and its possible relationship to the decline in N2 fixation activity. Nitrogen fixation (u moles pl'lhr’l) Dry weight (g) 28 7C)“ 6C>‘ 4C)“ EK)‘ 20‘ 10' I I U U . V 25 30 35 4O 45 50 55 (SO 65 7O 75 80 85 r F I W V ' ‘ Days after planting Figure 2. Nitrogen fixation (a) and ontogenetic distribution of dry weight (b): total (T); leaves (L); stems (5); flowers (F); pods (P); and nodules of the cultivar 72 VUL 26689. so so 20- I 20 10~ - 10 4140‘ ~140 130- 130 120' “120 110' ”110 I ) 1(X)” " 100 >3; _so- - so J 80- W333}? - so Nitrogen fixation 1hr.l) - q & (u moles p Dry weight (g) 110 ' 1(X) 90I 80 '70 EN) .40 30I 2C) 10 29 . ldégg p ” 100 8C) \\ M57.“ N ...-u“. “rd-2.1%-?“ "I I I r I I l r 1 1 F I 25 30 35 4O 45 5O 55 60 05 7O 75 80 85 Days after planting Figure 3. Nitrogen fixation (a) dry weight (b) of vegetative (V) and, nodules (N) of the cultivar and ontogenetic distribution of and, reproductive (RE) structures 72 VUL 26689. 20 10 10 3O Dry weight (g) w V j 1 1 W 1 U T ' 1 i I fu(x) IF I - , f . 1 1 I i I w 1 1 2 3 4 5 6 7 8 9 10 . Harvest number (t) Figure 3a. Ontogenetic distribution of stem dry weight in the variety 72 VUL 26689. Values shown are derived from cubic polynomials. flection points (IF) and maxima (M) are shown. In- Nitrogen fixation (u moles pl-lhr-l) Dry weight (g) .a C) ..L N C .5 ..IL 0 100 9C) 80 70 6C) 40 2C) 10 31 p-w--~*m-.——--~—.- ...- - .. P ’ I I . V N ‘x I I , 4" u o A I) U L--:1_l_l,llr .II I h \. 1.4 ..n. ) ilk] T— . I j '1 25 80 35 4O 45 Days after planting 100 9C) '8C) 70 6C) 50 4C) 3C) 2C) 10 Figure 4: Nitrogen fixation (a) and ontogenetic distribution of dry weight (6): total (T); leaves (L); stems (5); flowers (F); pods (P); and nodules (N) of the cultivar Porrillo Slntetlco. 50 40. 30- 20- 10« 15' 10- -15 . -20 ~10 Dry weight (g) ffix) I I I U I I I I I I 1 2 .3 4 5 6 7 8 9 10 Harvest number (t) Figure ha. Ontogenetic distribution of leaf dry weight in the variety 72 VUL 26689. Values shown are derived from cubic polynomials. In- flection points (IF) and maxima (M) are shown. Nitrogen fixation hr~l) . ‘1 1 (u moles pi Dry weight (g) 33 3 2C) ~ b 2C) 10 -\ «'2‘» . 1O . - -."i..s“..:€:"' ' -\ / . '_i.!-'__‘:_ b 100' b 100 90’ " 90 80“ r 80 7o - * 70 6C)‘ ‘ 6C) 50" ' 50 40" “ ‘40 5K)" ~ :30 2C)“ - :20 ‘IO ' - 10 .-—..'*V W1 I T ‘1 I I I V U r 1 25 30 35 40 45 5O 55 60 05 7O 75 00 85 Days after planting Figure 5. Nitrogen fixation (a) dry weight (h) of vegetative (V) and, nodules (N) of the cultivar and ontogenetic distribution of and, reproductive (RE) structures Porrillo Sintetico. 34 Stem weight was found to increase continuously until 81 dap (harvest 9), subsequently declining at harvest 10 (88 dap). Correspon- ding values and Analysis of Variance are given in Appendix Table 3. Cubic polynomials were fitted to the dry weights of leaves and stems and the results are shown in Table 2. Figures 3a and 4a show the plots of the relative growth rates of these two organs (first derivative of the polynomials) and the plots of acceleration of dry weight (second derivative). It is interesting to observe that leaf dry weight in the cultivar 72 VUL 26689, show a very early inflection point at 37 dap (2.75 harvests) in relative growth rate. This is taken as evidence that allocation of resources favors these structures early on so that the photosynthetic system be established to provide the future demands of other processes, such as nitrogen fixation. Stem dry weight was found to show an inflection point at 60 dap (5.97 harvests) in the same cultivar. As is later shown in this chapter, at 60 dap temporary storage of starch in the stem is initiated at this time. Beyond 60 dap the acceleration of dry weight in stems decreases and clearly reflects the onset of starch storage mentioned above. Also, maximum nitrogen fixation rates occurred at this time. The relative growth rate of leaf dry weight became zero at 70 dap (7.43 harvests), while stems exhibited a zero relative growth rate at 9.99 harvests. Similar results were obtained with the remaining cultivars. Figures 3 and 5 illustrate the dry weight allocation pattern to vegetative and reproductive structures, nodules, and their correlation in time with N2 fixation rates. Corresponding values and Analysis of Variance are given in Appendix Tables 5 and 8. Reproductive structures include flowers, pods and the rachis of the racemose inflorescence. 35 Table 2. Cubic polynomials fitted to stem dry weights (A) and leaf dry weights (B) and harvests (t, independent variable) of four dry bean cultivars (C). First and second derivatives, maximums, and points of inflection are shown. A. Stem dry weight R2 01 - 8.215708 - 8.317555 c + 2.567223 :2 - 0.143593 :3 0.958 02 = 4.172883 - 3.495695 6 + 1.288020 :2 - 0.062857 :3 0.991 03 = 3.592725 - 3.208141 t + 1.298141 :2 - 0.072893 t3 0.984 C4 = 5.664083 - 5.333215 6 + 1.843301 :2 - 0.104465 t3 0.988 First derivatives f'(x)=Ol c1 = -0.430779 :2 + 5.13445 t - 8.317555 9.9 02 . -0.188570 :2 + 2.57604 t - 3.495695 12.13 03 - -0.218680 :2 + 2.59198 6 - 3.208141 10.45 04 = -0.313400 :2 + 3.68660 t - 5.333215 10.07 Second derivatives f"(x)=01 c1 . -2t + 11.94 ' 5.97 02 . -2c + 13.66 6.83 03 = -2c + 11.85 5.92 c a -2c + 11.76 5.88 36 Table 2. (cont'd.) B. Leaf dry weight R2 01 - -7.556008 + 7.92070 6 + 1.523418 :2 — 0.184524 t3 0.936 02 - -5.355783 + 6.94349 t + 1.097879 :2 - 0.118747 :3 0.945 03 = -9.165492 + 11.4805 c - 0.142661 :2 - 0.052869 t3 0.847 04 = -7.475542 + 8.89930 6 + 1.007260 :2 — 0.133980 t3 0.931 First derivatives f'(x)=01 c1 - 0.553572 :2 + 3.04684 c + 7 92070 7.31 c2 - -0.356242 :2 + 2.19576 c + 6.94349 8.46 03 - -0.158606 c2 - 0.28532 c + 11.4800 7.65 04 - -0.401940 :2 + 2.01443 6 + 8.89900 7.84 Second derivatives f"(x)=01 c1 - -2c + 5.50 2.75 02 - -2t + 6.16 3.08 c3 - -2c - 1.80 0.89 04 - -2c + 5.01 2.51 1: Values of maximums and inflection points are given as harvests, i.e. 9.9 harvests. Cultivars: Cl - 72 Vul 26689, C = ICA Pijao, 2 C3 - Nep-Z, C4 = Porrillo Sintetico 37 Declines in N2 fixation rates and nodule dry weights coincide with the initial stages of reproductive development. It is important to note that at the time fixation declines, leaf and stem dry weight increases are of a higher magnitude as compared to the increases observed in the reproductive sturcutres. This has lead this author to entertain and evaluate the possibility that it is not ”competition" by reproductive organs which explains the observed drop in fixation for the dry bean cultivars under consideration. The allocation 2: soluble sugars and starch Carbohydrate levels (ethanol—soluble sugars and starch) were determined in all organs previously mentioned, with the exception of rachis and flowers. Chemical determinations were made from 39 dap (harvest 3) for nodules and from 53 dap (harvest 5) for all other organs or structures, until 88 dap (harvest 10). Figures 6, 8, 10, and 12, illustrate the values obtained in terms of total content of carbohydrate per plant. Corresponding values and Analyses of Variance are given in Appendix Tables 12, 14, l6, 18, 20, 22, 24, 26, 28, 30, 32, and 34. Figures 7,9,]Jq 13,illustrate carbohydrate levels expressed as concentration, i.e., mg of carbohy- derate/g dry weight. Corresponding values and Analyses of Variance are given in Appendix Tables 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35. Both kinds of information are included since they represent complementary aspects that relate directly to the hypothesis under consideration. Figures 6 and 7 and, 8 and 9, show the trends of the carbohydrate levels found in nodules, stems, and leaves for 72 VUL 26689 and Porrillo Sintetico, respectively. Total content of starch and sugars 2n: nodules is exhibited by Porrillo Sintetico, NEP-Z, and ICA Pijao, xation .1 hflhl) Nitrogen f1 (n nudes pi "111191-0713 of carbohydrateKnodu'les) .180 ‘ 38 3° ‘ a. . .. 80 :Jln 7 320 “ {mo~ * 280.‘ 200 ‘ 240 ‘ 220 ‘ 200 ' 160 “ 1 ' 400 140 ' Milligrams of carbohydrate (leaves-stems) 120 ‘ ’ 3 r 300 loo ‘ 1 I, r 250 80‘ ’ #200 . ' I to I ‘ '150 40 ' . L100 ’0. p” ‘s N ‘ 50 ---..v’-"~~- N I ' ' ' ' U T V I I 1 85 88 88 46 68 60 67 74 81 88 ' ’ Dnyl after planting ' Figure 6.‘ Nitrogen fixation (n) and content of ethanol-soluble carbohy— drates (broken line) and starch (solid line) (h) in leaves (L), stems (S), and nodules (N) of the cultivar 72 VUL 26689. A f: H O I H P U .C t‘.‘ r-i X I ad '4 u 4 n. C (D 0 0.) {-0 H O O H H u H :1 Z V "111197-811: ‘of carbohydrate / g. dry weight 39 25 32 39 46 53 60 07 74 81 . Days after planting 30 ‘ 20 l 10 ‘ 0 "W' T 1’ ’V‘ 1 " r 1 “” r" "" i “'“"l‘ I“- 450 ‘ 425 a 400 - 375 ' 350 325 ' 300 ‘ 275 ' 250 ' 225 ‘ 200 3 S . I‘- r '3." o ‘ 175 .1 \‘\ I, ‘I \\ ‘ , ,’ \\ .. 15° .. , \h ’g’ ‘ \ ’S In: \ a 125 - ‘ " \\ ' " \’ ‘v’ .100 ‘ ' , I: 76 J “I; ’l‘*~~~ I’A\‘s~_-- ”’7'." 60 . '(” .-~’ N .26 4 I r I I 1 I r r v 1 88 Figure 7. Nitrogen fixation (a) and concentration of ethanol-soluble carbohydrates (broken line) and starch (solid line) (b) in leaves (L), stems (S), and nodules (N) of the cultivar 72 VUL 26689. 250 225 200 150 125 100 50 25 Milligrams of carbohydrate I plant 340 320 .300 280 260 240 220 200 180 160 146 120 100 88 20 40 r ' Y Y I j 1 1 ' 25 32 39 46 53 60 67 74 81 . 88 Days after planting Figure 8. Content of ethanol-soluble carbohydrates (broken lines) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar 72 VUL 26689. 340 320 300 280 260 240 220 _—200 180 160 _ 140 120 100 80 60 40 20 41. Milligrams of carbohydrate /, g.- dry Weight .350 . > > t 350 325 q _ 325 300 . 300 275 4 4 275 250 .’ _ 250 225 _ 225 200 . . 200 175 - 175 150 . . 150 125 . _ 125 100 . L 100 75 . 1 75 ... .50 25 - » 25 25 32 39 46 53 .60 67 74 81 ‘ 88 Days after planting Figure 9. Concentration of ethanol-soluble carbohydrates (broken line) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar 72 VUL 26689. lhr‘l) Nitrogen fixation (u moles pl' Milligrams of carbohydrate (nodules) '280 ' 42 CD 0 a N o l H O J O 200 ' 240 ‘ 220 ‘ 200 ' 180 ‘ 160 ‘ 140 ‘ 120 . 100 ‘ 80 ‘ 60 - 40 . 20 . a -~—--' I 1 I . 53 60 _ 67 Days after planting Figure 10. Nitrogen fixation (a) and content of ethanol-soluble carbohydrates (broken line) and starch solid line) (b) in leaves (L), stems (S), and nodules (N) of the cultivar Porrillo Sintetico. 400 350 b 300 250 200 150 100 Milligrams of carbohydrate (leaves-stems) ‘Ffi-h C IN] ‘lll-I! \ 1" ‘Ilullllllci I‘ll. II I! .‘ ) i‘ Nitrogen fixatic ’1 (0 moles pl h - v- I Milligrams of carbohydrate / g. dry weight. 43 .,.__...__......___.._.--.--_...........-_..-..._-....-wn - ._. . ......m... . --....mwn ...- ..-..-fi 30 i -_i a 20 ‘ 1o -. 0 l”" I I I“ 225 ‘ h 200 ‘ 175 “ 150 ‘ 125 ‘ 100 ’ 75 ' 50 ‘ 25 ‘ 0 I l I I T’ I 1 I ’1 II c . 200 ' . 175 “ 150 ‘ 125 ‘ 100 ' ___;‘-§ r‘bl 75 ' /"\ r"~\ I] \\\ F--~-.._——” ‘~~___— I \ 50 J ,{’ '/ N ’I 25 o- I I I I I I I r I I‘— 25 32 39 46 53 60 07 74 81 88 Days after planting Figure 11. Nitrogen fixation (a) and concentration of ethanol-soluble Ii(l 20 10 225 200 100 carbohydrates (broken line) and starch (solid line) (b, c) in leaves (L), stems (S), and nodules (N) of the cultivar Porrillo Sintetico. N b C N N C 200 180 160 140 120 Milligrams of carbohydrate / plant '8' 80 60 40 20' 44 I U I I I *1 T I I I 25 32 39 46 53 60 67 74 81 88 ' Days after planting Figure 12. Content of ethanol-soluble carbohydrates (broken lines) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar Porrillo Sintetico. 320 300 ' 280 260 . 240 . 220 200 180 160 140 120 100 80 60 40 20 Milligrams of carbohydrate / g. dry weight 350 . 325 '300 . 275 250 225 200 175 150 125 . 100 . 75 - 50 25. 45 I “I I I I I I I I Iv 25 32 39 46 53 60 67 74 81 88 Days after planting 7 350 325 300 F- 275 250 225 ~ 200 175 150 125 100 75 50 25 Figure 13. Concentration of ethanol-soluble. carbohydrates (broken line) and starch (solid line) in primary roots (I), secondary roots (R), and pods (P) of the cultivar Porrillo Sintetico. 46 Table 3. Linear regression equations predicting nitrogen fixation rates for a given level of nodule soluble carbohydrate, and a given level of nodule starch. Cultivar 72 VUL 26689 ICA Pijao NEP-Z Porrillo Sintetico Linear regression eguation (u moles C .084 .477 .178 .566 .822 .583 .923 .350 -1 2114 pl X * + 1 962 1 . a X + 2.055 2 X1 + 2.314 X2 - 2.317 X1 - 0.821 X2 - 2.206 X1 - 2.608 X2 - 2.909 hr.-l 0.715 0.717 0.850 0.912 0.858 0.913 0.712 0.619 * X = mg. nodule soluble carbohydrate/plant 1 X = mg. nodule starch/plant 2 47 as shown in Appendix Table 14, is achieved in the cultivar 72 VUL 26689. The sites of starch accumulation in nodules are illustrated in Figures 18, 19, 20, 21, 22, 23. Starch accumulation was found to be restricted to the uninfected cells of bean nodules. Similar observations have been reported by (McCoy, 1929 and Allen and Allen, 1958). Linear regression analysis between fixation activity (umoles l hr—l) and carbohydrate level in the nodules were performed C2114 pl- and the results are shown in Table 3. A very close correlation among these values is seen to exist. It is important to note that total starch and sugar content follow the nodule mass values in synchrony and that the loss in dry weight is evidently followed by a loss in carbohydrate. The results illustrated in Figures 6 and 10 clearly indicate that at harvest 6 (60 dap), an opposite trend in carbohydrate content of leaves and stems versus nodules is established. Sites of starch storage in stems are illustrated in Figures 24, 25, 26. Starch grains were found to be primarily restricted to parenchyma cells of the axial rays of secondary xylem. Soluble sugars and starch content significantly increase in stems and leaves. Soluble carbohydrate content reaches a maximum level in leaves at 67 dap. (harvest 7), while soluble carbohydrates of stems continue to increase up to 88 dap (harvest 10). Starch in leaves increases up to 74 dap and subsequently rapidly decreases. The trend associated with starch increases following an exponential pattern from 60 dap to 81 dap appears important. This indicates that available sinks (i.e., reproductive sinks) have not reached the stage of develop— ment at which carbohydrate and nitrogen demand is maximum. By 88 dao, starch content has decreased in the stems. From this point onwards, 48 both temporarily stored carbohydrates and photosynthetic assimilates are being directed towards the seeds. This pattern is consistent in qualitative terms for all four varieties. Figures 7 and 11 illustrate the patterns in the concentration of soluble sugars and starch for 72 VUL 26689 and Porrillo Sintetico. Two trends, in particular, can be discerned in these figures: 1) the concentration of starch in stems is the largest among all structures studied and increases in the way indicated above, and 2) nodules contain large concentrations of carbohydrates, particularly starch. The latter figures also illustrate that nodule starch concentration continuously decreases after reaching a maximum level in harvest 5 (53 dap). This maximum level of starch concentration is seen to occur prior to the time of maximum fixation levels in all varieties. The decrease in the concentration of nodule starch may be an indication that this polysaccharide is used as an indirect energy source for growth and N2 fixation. The concentration of soluble sugars in nodules has been found to fluctuate only within relatively narrow ranges in all varieties. A consistent rise in concentration in harvest(5(60 dap), has been observed. It is important to notice, too, that even after the period of maximum N2 fixation activity, nodule soluble sugar concentration remains relatively constant, but nitrogen fixation subsequently declines. This clearly indicates a nodule population effect is responsible for the high fixation rates that occur during harvests 5 and 6, and that the drop in fixation follows a declining nodule population. Figures 8 and 9 illustrate the corresponding values for soluble sugar and starch content and concentration for 72 VUL 26689 in the underground portion of stems ("primary roots"), the main root system 49 ("secondary roots"), and pods. Trends in both soluble sugars and starch clearly indicate that these compounds continue to accumulate in the root system up to 81 dap (harvest 9). This trend is consistent with the observed increase in soluble sugars and starch found to be exhibited by nodules at this point and with the small burst of activity in N2 fixation. These observations would also appear to indicate that carbohydrate is not limiting N2 fixation. Similar trends for Porrillo Sintetico are illustrated in Figures 12 and 13. Corresponding values and Analyses of Variance for these figures are given in Appendix Tables 16 through 23, and 32 through 35. These trends were also found in NEP—Z and ICA Pijao. Soluble sugar and starch content of pods is also illustrated in the latter figures. These carbohydrates are seen to increase expon— entially from 74 dap onwards. This increase coincides with a buildup of carbohydrates in stems and leaves. Soluble sugars in pods are higher than starch content of pods. This relationship clearly indi~ cates that carbohydrates are primarily being used in pods at this stage for growth and that reproductive structures have not initiated their characteristic storage phase. This also probably indicates a relatively low absolute demand for photosynthetic and temporarily stored reserves. Distribution gf_organic nitrogen The distributions of total organic nitrogen and organic nitrogen concentration in "primary roots", "secondary roots”, stems, leaves, and pods, are shown in Figures 14 and 15 for the cultivars 72 VUL 26689 and Porrillo Sintetico. Corresponding values and their Analyses of Variance are given in Appendix Tables 36 through 48. Total nitrogen content for the summation of nitrogen content of roots, stems- and tion :ixa Nitrogen Q hx-*) OD C 1 (u moles p1- .t: O) 8'— WE (mg / 9 dry (mg 7' pl ant) Organic nitrogen 10 10 100 140 120 100 80 60 40 20 50 “--uav4.———_——I—v 0..-. w“~ofio-*-.~-o ...—.....- - / / ‘\ *“wm- / ”"*—1 '"° 'm’T“-——"I"" "'1 “'W‘ T” -"'l"'"'""‘ T‘“_”’ 'T‘"""""l ‘"' "' Th” h ..1 “‘“\ ~.\‘ SAR ‘ttr-J .1 —"-.-_ ----_‘-_-\ ll, ‘\‘ \‘~. 4 s\‘ SN ”"N‘ -..__ _,___....-. MN 1 TAR C 25 32 39 40 53 00 67 74 81 88 Days after planting Figure 14. Nitrogen fixation (a), organic nitrogen concentration (b) and content (c) of leaves (LN), stems (SN), primary roots (TAR), secondary roots (SAR), and pods (PN) of the cultivar 72 VUL 26689. 30 20 lo 100 140 120 100 80 60 40 20 Nitrogen fixation 121-1 hit—1) (u nxfles (mg / g dry weight) (m9 / plant) ‘ Organic nitrogen a. O 10 50 40. 20 10 160 140 120 100 80 00 40 20 51 0 r‘ a g ”‘5. ...1 I I I I I -1 1w - —_.1 ' __--.___'__..--._ b _ LN t q \ >- .\ _ 1-- SAR -I \— ’’’’’’’’’’ ~ ~ ~— ————————————————— L. '4 \ >- IAR C .1 i. SN ’vfi". ..--'- . ,5» P . . ”.--v—r - IAR ~ I I j. ' I 1 1 I 1 U 25 32 39 40 53 00 07‘ 74 81 88 Days after planting Figure 15. Nitrogen fixation (a), organic nitrogen concentration (b) and content (c) of leaves (LN), stems (SN), primary roots (lAR), secon— dary roots (SAR), and pods (PN) of the cultivar Porrillo Sintetico. 40 30 20 10 160 140 120 100 80 00 40 20 52 leaves, is shown in Table 4 and in Appendix Table 44. Total organic nitrogen per plant increases up to 81 dap (at harvest 9), subsequently decreasing. The major portion of the nitrogen present in vegetative tissues is accounted for by leaf nitrogen. The pattern exhibited by the concentration of organic nitrogen in ”primary roots". stems and leaves is, in general, a similar one. This pattern is more easily observed in the corresponding tables of the Appendix. It is observed that nitrogen concentration progressivley declines up to about 88 dap. Slight increases during the periods of maximum fixation rates (60 and 67 dap) are exhibited by leaves and stems. Cubic poly- nomials were fitted to the total nitrogen content of leaves and stems throughout the period studied. Varieties were pooled in this analysis. Curves are obtained for total nitrogen in these structures very similar to the ones obtained when the dry weights were fitted. Fitted curves and the corresponding polynomials are given in Figures 16 and 17, and in Table 5 respectively. An inflection point at 2.68 harvests was found in leaves. A maxima, as indicated by the first derivative going to zero and the sign of the second derivative, was found at 7.29 harvests. Stem total nitrogen exhibited an inflection point at 5.38 harvests and a maxima at 9.34 harvests. The values exhibited by leaves can possibly be taken as evidence that nitrogen is no longer accumulated beyond the 7.29 harvest point and in fact, it may indicate that leaves may be starting to mobilize nitrogen out of them. This view is supported by the seasonal profiles of N2 fixation. The level of organic nitrogen in pods is seen to increase from 74 dap to 88 dap, but the level of nitrogen in these structures is low compared to the level of nitrogen usually found in seeds. which is about 38.4 mg N/g. dry weight if one assumes a mean value of protein 4‘0 30 20 '10 20 10 -20 10 53 l .fll(x) Harvest number (t) Figure 16. Ontogenetic distribution of stem nitrogen. Values shown are derived from cubic polynomials. are shown. Inflection points (IF) and maxima (M) VA “3.4..— 40 -2s 20 '10 ~20 200 160 .~ 120 3 80 3 40 30- 20 10. 54 mngl fn(x) IF 6 7 8 9 10 Harvest number (t) Figure 17. Ontogenetic distribution of leaf nitrogen. Values shown are derived from cubic polynomials. maxima (M) are shown. Inflection points (IF) and . . {N 1\ on! n e—fliph. I u .nA-nsF-n- . a. '1' 1 .. 4 .. . .u a. I a Each H nwflh< . o--h ...) u is d as: Fauna-Z \n v 1- . . 1 < .. ~ ‘ P u Furs: viuan I H «vanesww In ~43 Autyd>a~.-— ...-.3 ass-:96 of. a... .. . . . a in— -.Au.u he. nu—Hrn.v>1~q-~ >~ vu..u.,.v3 Mean _u-...v-~uuimu .....vh .un.» u..., I-V QtIUL-c nus-.1. ,unk It). unauoox.v> in. nun-vh-IGNU annusw$aslhiuufih vvh-nsnxiinav 1’ N Q! . \‘ s...‘ I. 2u~o 55 wswusoau nouns when H 6H.maa mm.a~a aq.oma ma.ama mm “a.m2~ ~m.o~a 4~.mHN mm.maa mm m~.maa ma.mHH om.maa ~o.m~a as an.aaa aa.ama oa.oma ao.m¢a as mo.maa Hm.~qa ma.aoa m~.oaa co H~.m~a a~.oa ma.aoa on.a~a mm em.aaa ~8.maa ma.oaa a~.oaa as No.~o ao.oo om.wo ma.~e on ~o.ae o~.mm an.~m no.8n an H~.a~ mo.o~ me.a~ oa.m~ ma Ausoao\z may AusmHo\z wav Ausoao\z way Ausoao\z wsv (Hanovv coaumuaam oHHaoooa Nunez oafiaa aoH awoeu a=> as mass umu>uam um>wuasu .qo wanes xwvsooq< ow vousoooum ma oosmaum> wo maohams< .muo>wuaso soon mum soon mo muoo>umn saxooa Howusosvoo sou mo Aoo>moa was .maouo .ouoouv mousuosuum e>aumuowo> mo usousoo sowouuws oasowuo .4 edema Table 5. 56 Cubic polynomials fitted to organic nitrogen content of stems (A), leaves (B) and, harvests (t independent variable) of four dry bean cultivars. Corresponding 95% confidence upper and lower limits (CL) are given. A. Stem nitrogen: R2 = 0.765 2 3 N content==10.55269050-9.30912632 t+-3.38087446 t - 0.20838879 t N content Harvest time1 Lower 95% CL (mg/plant Upper 95% CL 1 0.042 4.416 8.790 2 1.144 3.791 6.438 3 4.675 7.427 10.179 4 11.401 14.073 16.745 5 20.114 22.480 24.846 6 29.039 31.397 33.763 7 36.902 39.574 42.245 8 43.008 45.761 48.513 9 46.059 48.706 51.353 10 42.786 47.160 51.534 Table 5. (cont'd.) B. N content = 13. 81823221 + 27. Leaf nitrogen: 2 = 0. 57 635 369l3lll.t+-2.90227790 t + 0.38194592 t N content 2 3 Harvest time1 10 Lower 95% CL mg/Elant -4.704 16.071 36.902 49.474 71.026 84.097 104.958 117.651 136.603 147.841 161.140 172.378 176.277 188.970 182.253 195.324 176.578 189.150 147.379 168.155 Upper 95% CL 36.847 62.045 97.169 130.342 159.079 183.616 201.662 208.396 201.721 188.930 1: Harvest time (t) given as the harvest number in which samples were collected. 58 N in seeds of 24%. Increases in plant nitrogen beyond the point of N2 fixation decline are expected to be small compared to values that are present in the vegetative stage of the plants. This suggests that a major portion of seed nitrogen is derived from mobilization of the nitrogen present in leaves and stems. Evaluation of the original working hybothesis postulated on the basis of reported research requires that answers be provided to the following questions: 1) does a change occur in the pattern of distri- bution of carbohydrates to the nodulated system?, i.e., is parti~ tioning of assimilates altered between reproductive and vegetative structures?, and 2) is this change or alteration in the partitioning of carbohydrates correlated with the development of reproductive structures, with a decline in assimilates in the roots and nodules and with a decline in nitrogen fixation?. The approach to answering these questions is a sequential one in time and is as follows. Soluble carbohydrates and starch in nodules were shown to reach a maximum level at 60 dap in all four cultivars and subsequently declined. Since a similar pattern was exhibited by nodule dry weights the population level of nodules appears to be correlated with high nitrogen fixation rates in the period of 60 to 67 days after planting. Carbohydrate levels (starch and sugars), when expressed as concen- tration in the nodules, provide a different but complementary kind of information. It would be expected that because of the high energy requirements for N2 fixation, a higher concentration of soluble carbohydrate would be present during the maximum fixation period. The data collected during the conduct of experiments has failed to support this view as shown in Appendix Table 13. In fact, soluble sugar concentration remains relatively constant. This is taken as an 1'2 Cl CC 59 indication that these figures reflect the level of sugar concentraion required to maintain basal metabolism. This leads to the possibility that soluble carbohydrate translocated to the nodules is very rapidly used in the fixation of nitrogen and, consequently, the data obtained for soluble sugars reflects "basal metabolism status". Starch build» up in nodules has been shown to occur prior to maximum fixation rates, subsequently declining. It is suggested that this decrease in starch concentration is an indication that the products of hydrolysis of starch are being utilized as a supplement to the high energy require- ments that have to be met during the period in which maximum fixation rates of nitrogen occur. Most nodules senesce subsequent to the occurrence of the highest fixation rates. This view seems to be supported. Nodules are borne in larger numbers in the upper portion of the root system of bean plants. Senescence of nodules after the peak of fixation is associated with this major portion of the population of nodules present in the plant. One more aspect deserves considera- tion. It has been pointed out that lower leaf abscission occurs in dry bean plants between 67 dap and 74 dap, and this coincides with: l) the drop in nitrogenase activity; 2) the drop in nodule dry weight; 3) a decline in total sugars and starch; and 4) a decline in starch concentration, but not with soluble sugar concentration in nodules. This later aspect, no decline in soluble sugar concentration in nodules, is not surprising since nodules samples were not physically deteriora- ting. The evidence reported in the literature, and that presented in this thesis, relating an energy supply to nodules and their activity conflict in the interpretation of the sequence of events that leads to the tea nod dat C01 as: veg C01 OPT ca' 110 in St ni C0 60 the decline of this activity. The conflict of opinion resides in the reason(s) for the reduction in the supply of carbohydrates to the nodules. Several authors (3, 8, ll, 12, 13, 22) have suggested thatthe data presented by them indicate that such a decline in activity is a consequence of "competition from reproductive sinks” for photosynthetic assimilates. The data presented in this section indicates that vegetative organs, leaves and stems in particular, become temporary storage sites for carbohydrates. Hence, in all varieties under consideration, carbohydrate levels in stems and leaves show an opposite trend to that found in nodules. It has also been shown that carbohydrate levels in the underground structures of bean plants do not decrease in this period, they in fact show a tendency towards increasing. Since, reproductive structures, during the period studied, represent a small sink for carbohydrates and organic nitrogen, it is doubtful that they reduce N fixation primarily by 2 competing for carbohydrates. Figure 18. Figure 19. Figure 20. 61 Cross section of a Phaseolus vulgaris L. nodule at 53 dap with central bacterial cells (B), peripheral vascular bundle (V), cortex (C) and, uninfected cells (U) containing compound amiloplasts (A). Periodic Acid Schiff - stained section X40. Cross section of a Phaseolus vulgaris L. nodule at 53 dap illustrating crenula (CR), vascular bundle (V), bacteriod containing cells (B), and uninfected cells (U) containing compound amyloplasts (A). Periodic Acid Schiff - stained section X400. Cross section of a Phaseolus vulgaris L. nodule at 53 dap with peripheral vascular bundles (V), bacteriod containing cells (B), uninfected cells (U) and, com— pound amyloplasts. Toluidine Blue-stained section. X100. 62 1-—— Figure 21. Figure 22. Figure 23. 63 Cross section of a Phaseolus vulgaris L. nodule at 53 dap illustrating bacteriod containing cells (B), nucleous (N), and uninfected (U) cells containing compound amyloplasts (A). Mercuric-Bromphenol Blue - stained section. 560x. Cross section of a Phaseolus vulgaris L. nodule vascular bundle at 53 dap with endodermis (E), casporian strips (CS), xylem (X), phloem (PH) and, compound amyloplasts (A). 800X. Bacteriod containing cells (B) and uninfected cells (U) with nucleous (N), nucleolus (ns) and, compound amyloplasts (A). 850x. 64 Figure 24. Figure 25. Figure 26. 65 Cross section of stem at third node level illustra- tion amyloplasts (A) in secondary parenchyma cells. Phase-contrast photograph. 400X. Cross section of stem at the third node level illustra- ting amyloplasts (A) in parenchyma cells of the pith. Phase-contrast photograph. 4OOX. Cross section of stem at the third node level illustra- ting amyloplasts (A) primarilly located in the porenchyma cells of rays of secondary xylem. Periodic Acid Schiff - stained section. 140x. 66 10. 67 REFERENCES Allen E. K. and O. N. Allen. 1958. Biological aspects of symbiotic nitrogen fixation. In "Encyclopedia of Plant Physiology." F. C. Steward, ed. Academic Press, N.Y. Vol. 8. pp. 48—118. Dart, P. J. 1974. Biological nitrogen fixation. Eighth meeting of the consultative group on international agri- cultural research. Technical Advisory Committee., Washington D. C. Dart, P. J., J. M. Day, A. R. J. Eaglesham, P. A. Huxley, F. Minchin and R. J. Summerfield. 1976. In "Joint FAO/IAEA Coordinated Programme on the Use of Radioiso- topes in Fertilizer Efficiency Studies in Grain Legumes." Vienna, Paris. Day, J. M. 1972. Studies on the role of light in legume symbiosis. Ph.D. thesis. University of London, England. Feder, N. and T. P. O'Brien. 1968. Plant microtechnique: some principles and methods. Amer. J. Bot. 55: 123-142. Gibson, A. H. 1976. N2 input into crops. 1. Obligatory Symbiosis. (Limitation to dinitrogen by legumes). In "Proceedings of the 1st. International Symposium on Nitrogen Fixation." W. E. Newton and C. J. Nymann, eds. Washington State Univ. Press. Pullman, Wash. Vol. 2. pp. 400-428. Graham, P. H. 1975. In "Annual Report." Centro Internacional de Agricultura Tropical, CIAT, Cali, Colombia. pp. 024—029. Hardy, R. W. F. and U. D. Havelka. 1975. Photosynthate as a major factor limiting N2 fixation by field grown legumes with emphasis on soybeans. In "Symbiotic Nitrogen Fixation in Plants." P. S. Nutman, ed. Cambridge Univ. Press. New York. pp. 421-439. Hardy. R. W. F., R. C. Burns, R. R. Hebert, R. D. Holsten and E. K. Jackson. 1971. Biological nitrogen fixation: A key to world protein. In "Biological Nitrogen Fixation in Natural and Agricultural Habitats". Lie, T. A. and E. G. Mulder, eds. Plant and Soil: 561-590. Hardy, R. W. F., R. D. Holsten, E. K. Jackson and R. C. Burns. 1968. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43: 1185-1207. ll. 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 68 Lawn, R. J. and W. G. W. Kurz. 1974. Symbiotic nitrogen fixation in soybeans. I. Effect of photosynthetic source—sink manipu- lations. Crop Sci. 14: ll-l6. Lawrie, A. C. and C. T. Wheeler. 1974. The effects of flowering and fruit formation on the supply of photosynthetic assimilates to the nodules of Pisum sativum L. in relation to the fixation. of nitrogen. New Phytol. 73: 1119—1127. Lawrie, A. C. and C. T. Wheeler. 1974. Nitrogen fixation in the nodules of Vicia faba L. in relation to the assimilation of carbon. New Phytol. 74: 429-436. Lawrie, A. C. and C. T. Wheeler 1973. The supply of photosynthetic assimilates to nodules of Pisum sativum L. in relation to the fixation of nitrogen. New Phytol. 12: 1341-1348. Mague, T. H. and R. H. Burris. 1972. Reduction of actetylene and nitrogen by field grown soybeans. New Phytol. 71: 275-286. Mazia, D., P.A. Brewer and M. Alfert. 1953. The cytochemical staining of protein with mercuric bromphenol blue. Biol. Bull. 104: 57-67. McCoy, E. F. 1929. A cytological and histochemical study of root nodules of the bean, Phaseolus vulgaris L. Centbl. Bakt. II. 79:394-412. O'Brien, T. P. and M. E. McCully. 1969. Plant Structure and Development. The Macmillan Co./Collier-Macmillan, Ltd. London. pp. 114. Ruthman, A. 1970. Methods in cell research. Cornell Univ. Press., Ithaca, New York. pp. 368. Pate, J. S. 1958. Nodulation studies in legumes. I. The synchromization of host and symbiotic development in the field pea, Pisum arvense L. Aust. J. Biol. Sci. 366-381. Pate, J. S. 1958. Nodulation studies in legumes. II. The influence of various environmental factors on symbiotic expression in the vetch (Vicia faba L.) and other legumes. Aust. J. Biol. Sci. 11: 496—515. Quebedeaux, B. and R. W. F. Hardy. 1975. Reproductive growth and dry matter production of Glycine max (L.) Merr. in response to oxygen concentration. Plant Physiol. 55: 102-107. Quebedeaux, B. and R. W. F. Hardy. 1973. Oxygen as a new factor controlling reproductive growth. Nature. 243: 477-479. Yoshida, S., D. A. Porno and J. H. Cock. 1971. Laboratory Manual for Physiological Studies of Rice. The International Rice Research Institute. Los Banos, Phillippines. pp. 61. CHAPTER 3 EFFECTS OF CARBON DIOXIDE ENRICHMENT ON SYMBIOTIC NITROGEN FIXATION IN PHASEOLQS‘VULGARIS L. Abstract Dry bean plants exposed to 1200 ppm carbon dioxide at various ffl""EF!i developmental stages exhibited higher nitrogen fixation rates, higher nodule fresh weights, higher total soluble carbohydrate and slightly higher organic nitrogen contents. Carbon dioxide treatment during the four weeks prior to reproductive growth, was found not to extend the duration, nor prevent the decline of nitrogen fixation. The view that the rate of nitrogen fixation pg£_§g is limited by photosynthate available to the entire symbiotic system is supported. 69 70 INTRODUCTION Studies conducted by several investigators have shown thatlegumes, such as soybeans, when exposed to carbon dioxide levels higher than those of air fix atmospheric nitrogen at higher rates than plants grown under prevailing carbon dioxide levels in the atmosphere (1, 2, 3, 4, 5, 6). This positive response has been attributed primarily to a reduction of photorespiration (l, 2, 3, 4, 5). The results reported in this chapter are the outcome of exposing bean plants to 1200 ppm of carbon dioxide during part of their vegeta- tive and reproductive period. Answers to the following questions were sought in this study: a) does carbon dioxide treatment result in a higher level of photosynthetic assimilates in treated plants?- b) are higher levels of soluble carbohydrates in nodules found in CO treated 2 plants?: c) are higher rates of nitrogen fixation attained through CO2 treatment?: and, d) is the seasonal decline in nitrogen fixationdelayed due to carbon dioxide treatment? MATERIALS AND METHODS Plants utilized in this study were grown on the experimental grounds of the International Center for Tropical Agriculture (CIAT) in Cali, Colombia in an area 14.5 x 28 m containing a sand-soil mixture (4:1 proportion). Fertilizers were not applied since results of soil analysis indicated adequate nutrient levels for plant growth. During the month of February, mean temperature at this location is 24 C. Prior to planting, soil temperatures measured at 10 cm depths varied from 25 C at 9 am to as high as 45.5 C by 3 pm. Due to the known 71 adverse effect of high temperatures on Rhiggbium survival and on nodulation a soil mulch of rice hulls (10 cm thick) was used. Mean temperatures at a depth of 10 cm varied from 24.4 C at 9 am to 28.9 C by 3 pm when the mulched was used. Porrillo Sintetico was the cultivar selected for this study. Seven double rows were planted with seed inoculated with the Rhiggbium phaseoli strain CIAT 57 on February 4, 1976. Seeds were wetted with a 10% aqueous solution of sucrose and subsequently the "peat"—(turba)~ based inoculum was applied to the wetted seeds. Inoculated seeds were allowed to dry in the sahde prior to hand planting. Planting distances were 35 cm between double the double rows and l m between adjacent double rows. Planting density was equivalent to 30 plants/m2. Carbon dioxide application The equipment utilized for delivery and metering of carbon dioxide is illustrated in the diagram shown below: é——- CO2 source I needle valve I tygon tubing 1 ' l I l . I blower 1)! J—q ,____F / air flow control air vent double rows of plants plastic chamber 72 The chambers used consisted of aluminum frames lined with heavy gage transparent plastic. Chambers were open at the top asillustrated. Two chambers, each supported on a wood frame, were supplied with C02 through a blower-plastic pipe assembly. Each chamber had a volume of l m3 (1000 l). The supply of air administered by the blowers was cali— brated to give a 0.5 change of air volume per minute per chamber. To provide a concentration of 1200 ppm C02, 100 m1 of carbon dioxide/5 seconds was metered to every pair of chambers used in this experiment. Carbon dioxide was metered with the aid of needle valves and a soap— bubble meter twice a day. Wind velocities at the plastic pipe openings were calibrated with an Alnor meter using tube number 602 for this purpose. Ten chambers were available for this experiment. Carbon dioxide treatments consisted of metering 1200 ppm C02 in the following manner: a) CO was applied for a period of two weeks (18—32 dap), using four 2 chambers for this purpose: b) CO2 was applied for a 4 week period (18-46 dap) utilizing four more chambers ' c) the chambers utilized for the 18-32 dap treatment were transferred on the thirty-second day after planting to the plants that received carbon dioxide from 32 to 46 dap and; d) the remaining two chambers were utilized to expose plants to a 4-week period (18-46 dap) but plants were not harvested until agronomic maturity was reached (95 dap on May 13, 1976). Appropriate controls not having been exposed to C02 were included for each treatment comparison. Flowering occurred at 32 days after plainting. Plants of the Porrillo Sintetico cultivar were known to flower at 35dap at this location. The scheduling of the carbon dioxide treatments was made to coincide with the expected decline in nitrogen fixation during the early periods of reproductive growth. 73 Acetylene — Ethylene reduction assays and sample preparation Acetylene - ethylene reduction assays were performed in the manner previously described in Chapter 2. Subsequent to completion of the assays the tops and nodulated roots were separated into component parts and dried as indicated previously. After drying, samples were weighed and subsequently ground for organic nitrogen determinations. Since four chambers were used per treatment, the plants of two chambers belonging to a different pair were selected at random for acetylene assays, dry weight and organic nitrogen determinations. The plants in the other two chambers were used for fresh weight determinations and soluble carbohydrate analyses. Each chamber contained 30 plants and the acetylene assays were performed with 5 nodulated roots/assay. Hence, six subsamples/chamber/treatment were available for each type of analysis indicated above. Fresh weight determinations were made immediately after removing the plants from the chambers and subsequently frozen at ~10 C. Organic nitrogen determinations These analyses were performed in the same manner indicated in Chapter 2. Determination of ethanol—soluble carbohydrates These analyses were performed in the manner indicated in Chapter 2 ‘with the exception of the extraction procedure. Nodules, roots and stems, were extracted with 80% analytical grade alcohol in a proportion of 1:10, i.e., 1 part of frozen tissue to 10 parts of 80% ethanol. Frozen samples were ground in a blender with ethanol and subsequently filtered under vacuum using hardened Whatman No. 1 filter paper. ‘Filtered samples were adjusted to known volumes and stored in a refrig— 74 erator in teflon—lined screw capped culture tubes. Chromogen develop- ment was performed following the procedure described in Chapter 2. RESULTS AND DISCUSSION Results concerning the effect of carbon dioxide on nitrogen fixation rates and nodule fresh weights are shown in Table 6. Corres— ponding analyses of variance are given in Appendix Tables 47 and 48. Statistical evaluation of the data in this section has revealed a problem of a small number of degrees of freedom for testing of hypothn eses. It is suggested that this be taken into consideration in the discussion that follows. Differences in nitrogen fixation rates at 18u32 dap were found not to be statistically significant, in spite of a 100-fold difference between carbon dioxide treated and untreated plants. Nodule fresh weights were found to be higher when plants were exposed to CO during 2 this period. No differences in nitrogen fixation rates or in nodule fresh weights during the 18—46 dap period were found between plants exposed to C02 and plants exposed to a normal concentration of carbon dioxide. The effects of carbon dioxide on fresh weight of roots, stems, leaves and pods are presented in Table 7. Corresponding analyses of variance are given in Appendix Tables 51, 54, 57, and 58. Root and stem fresh weights at 18-32 dap were found to be higher in carbon dioxide treated plants. A similar pattern was observed for leaf fresh weight but this difference is not statistically significant. For the period comprising 18 to 46 dap, no statistically significant differences were obtained for root and leaf fresh weights between C02 treated and untreated plants. Pod and stem fresh weights were found to be higher due to an increasing period of exposure to carbon dioxide. 75 Table 6. Effect of carbon dioxide on nitrogen fixation rates and nodule fresh weight of the dry bean cultivar Porrillo Sintetico. N2 fixation Nodule fresh weight CO enrichment (u moles 5 pl'1 hr-l) (g/54p1ants) 2 2 weeks 4 weeks 2 weeks 4 weeks - 30.67 2.40 1.0547 0.3747 + 63.87 ns 5.46 2.09091 0.5680 * + 2.92 ns 0.5255 ns * these plants received 002 enrichment only the last two weeks of the four week period. C02 treatments began 18 days after planting. 76 .wafiuamam umuwm sham ma amwon mucmaumouu moo .nofiuma xmmz q was we mxmoa osu umma was haso unmanofluam Nov vm>fiuomu madman mmonu m Hapmfl sufiflwnmpoua . ao.o v N Hm>6H suwflfinanoua . Ho.o v a. Hm>mfl susflwnmnoua . mo.o w « Hm>ma huHHHnmnoua I oa.o um massagmfiawam Huowumauwum on m: madman n\m s« H «soa.¢c ms nm.qwa ««HN.mm ma mo.m m+ o~.Hm lulu no.0wa m: Nc.cm Ne.cw NHw.om mm.oa «oo.m + wo.mo III: mo.NmH Nm.mm o¢.en m~.N~ NH.oH nw.o I mxoma a mxoms N mxmoa a mxooa N mxooz Q mxmma N mxmoa c mxmma N mvom mo>moa mamum muoom u:mfi:uauso Noo &wu Hugwmwa :wouh .ooaumusfiw oaaauuom um>wuaao smog muv onu mo uswwmz smouw won paw .uan03 ammuw mama .unwams smoum swam .uawamsinmmum use» so owfixowv conumo mo uuuwmm .n magma 1. Ci CC 5U! Ex; 77 Dry weights from a different set of plants but similarly treated are presented in Table 8. Corresponding analyses of variance are given in Appendix Tables 59, 62, 65 and 68. Exposure of plants to a higher carbon dioxide level than that of air did not have a positive effect on the dry weights of roots, stems and leaves at the time periods studied. Pod dry weight increases due to carbon dioxide treat- ment confirms the results obtained with of pod fresh weight. The effect of carbon dioxide on the content and concentration of soluble carbohydrates in nodules, roots and stems are presented in Table 9. Corresponding analyses of variance are presented in Appendix Tables 49, 50, 52, 53, 55, and 56. In the period comprising 18 to 32 dap, roots/soluble carbohydrate contents and nodules were found to be higher in plants exposed to carbon dioxide. In the longer time period (18-46 dap), soluble carbohydrate content in root and stem of CO2 treated plants was found to be higher than in the plants not exposed to carbon dioxide. However, no statistically significant differences can be associated with this response. Values concerning the concentration of carbohydrates indicate that the concentraion of soluble carbohydrate in nodule differ between CO exposed and unexposed 2 plants in an unexpected way. In this case, at 18-32 dap, soluble concentration of carbohydrates of nodules in CO ~treated plants is 2 lower than that exhibited by the nodules of plants not exposed to carbon dioxide. In contrast, during the same time period, the concen— tration of soluble carbohydrates in roots was found to be higher in COZ-treated plants than in plants not exposed to carbon dioxide. In a manner similar to that found in nodules, the concentration of soluble sugars in stems in this time period was found to be lower in plants exposed to carbon dioxide. At 18 to 46 dap, soluble carbohydrate root I I 1 1! >. . O x I . II I u I . I. II II IIIII.‘ til-r. .l‘f.\ .t Gilt h 1!!! >lht INAHRI UFH! u _ u. 3 hfiv Barre—r biz—fuse >an Emwum oUCMwunv: ufiL—J .n.ufive~ Fuflu mw—denufiFv Cnvn~u~nwrv “\C Una-1%.“ «m cm... mvflnnwrh 78 .msausmam wound mmmv ma comma mucoaumouu oo .coauma xmms c onu we mxoma 03u umma was mace unmasowuum Nov vo>fiwomu madman ommnu N Hm>oa xuaaanmnoua I OH.o um massagmwcwfim Hmowumwumum on ma madman m\w a“ H «smm.¢ m: mm.wN m: ON.oH m: mo.q N+ on.m IIII mm.oN ma mq.ma Hm.oa ms mn.m oa.m m: «H.N + Hm.e IIII mN.NN mm.NH mm.NH om.m oc.m NH.N I mxooa c axomz N mxmos q mxmoa N mama: q mxooa N @3003 q mxooa N mvom mo>moA mamum muoom uaoazUNuau Noo % H2363 mun .ucmeS mum wmoa .unwaws mum amum .unwama mum uoou so ovNNOHv nopumu mo uoommm .ooaumuaam oaawuuom um>HuH=o anon hum ens mo unwfima 5am mom was .w manna Table 9. 79 Effect of carbon dioxide on content and concentration of soluble carbohydrates of nodules, roots, and stems, of the dry bean cultivar Porrillo Sintetico. Soluble carbohydrate content CO2 enrichment (mg/5 plants) Nodules Roots Stems 2 weeks 4 weeks 2 weeks 4 weeks 2 weeks 4 weeks - 36.84 ---- 82.29 198.17 272.46 1,287.08 + 44.81* -—-- 116.56* 228.96 282.27ns 1,633.48 4.1 -—-- 221.31 ns 1,550. 70 ns CO2 enrichment Soluble carbohydrate concentration (mg/g fresh weight) Nodules 2 weeks 4 weeks Roots 2 weeks 4 weeks Stems 2 weeks 4 weeks 37.54 12.21 19.32 22.08* 13.00** 22.04 22.94 ns 12.11 17.20 9.21ns 19.63 17.20 ns ns no statistical significance at 0.10 - probability level * < ** IA 1 these plants received CO four week period. 0.05 - probability level 0.01 - probability level 2 enrichment only the last two weeks of the C02 treatment began 18 days after planting. ts du 01‘! 80 and stem levels were found to exhibit a trend of increasing concen~ tration with a longer period of exposure to carbon dioxide. Carbohy— drates in nodules were not analyzed during this period due to the smallness of the sample encountered. Table 10 shows the results obtained on the effect of carbon dioxide on organic nitrogen content and concentration of root, stem, leaf and pod. Corresponding analyses of variance are presented in Appendix Tables: 60, 61, 63, 64, 66, 67, 69, and 70. For the time period comprising 18 to 32 dap, nitrogen content of roots. stems and leaves was found not to differ significantly in a statistical sense between CO2 treated and untreated plants. A trend toward higher organic nitrogen content as a consequence of exposure to carbon dioxide in these structures can be surmised from the data shown. At 18 to 46 dap no statistically significant differences were detected between carbon dioxide treated and untreated plants in organic nitrogen content of roots, stems and leaves. A possible trend of response to carbon dioxide during 32 to 46 dap can be surmized from the results. In contrast, organic nitrogen content of pods was found to be signifi~ cantly higher in CO2 treated plants. A peculiar set of results was found for the treatment period 18 to 46 dap in terms of organic content. Root, stem, and leaf organic nitrogen were found to be higher than the untreated controls but pod nitrogen content was lower in CO2 treated plants. The effect of carbon dioxide on organic nitrogen concentration of roots, stems and leaves at 18-32 dap was found not to differ for the two treatments studied. In contrast, plants exposed to carbon dioxide during the 18 to 46 dap time period exhibited a peculiar response. The «Irganic nitrogen concentration of roots, stems, leaves and pods in 81 .wcHusmHm umumm mmmv wH amwon mucoaumouu Noo .vOHuma x003 snow wnu mo mxmms osu ummH mnu szo unmanUHuao Nou vm>Hmomu mucan 00039 H H6>6H suwflwnmpoua I Ho.o w «I H6>6H susawaunoua I mo.o w I Hm>mH auHHHnmnoua I 0H.o um muamonchHu HmUHumHumum on as ««MH.Nc IIII «on.Nm «xen.oH m: mm.NH H+ qw.~m IIII mN.mN ms oN.oq NN.¢H a: mN.NH wH.NH as no.mH + Ne.me IIII m¢.om MN.oq no.mH mw.oH m¢.NH we.qu msmum wuoom Austos hub m\wav ufi¢fi£0Huso Nou =0Huwuusmucoo mwwouUHs oHsumuo «soo.mam m: NN.¢Nm ms mN.moN ma No.Hm H+ Hm.msm IIII oN.mnN as HH.Nmm mm.mQN as nn.oe Hq.5¢ m: Nm.Nm + mm.mwN IIII HH.Nmo NN.NHm oN.HMN oo.mo HH.mq «N.Hm I axoos e «3003 N exams e axoua N 63003 c 63003 N 0x003 e axes: N avom mo>m0H mamum muoom Amuann m\wav unoEJUHusm Nov usouaou amuwuuH: oHnmmuo .muoou mo cowouuHs Uchwuo mo aoHusuucooaoo van usuuaoo so QVHNOHv nonuuu mo uowumm .OUHuuuch OHHHuuom um>HuH=o anon huv may «0 upon was .mo>uoH .mawum .OH oHan 82 carbon dioxide treated plants was found to be smaller than the corres— ponding untreated plants. When the values obtained for the concen- tration of organic nitrogen in control versus carbon dioxide treated plants are compared, the concentration of organic nitrogen in C02- treated plants was found to be lower. In Table 11, the results obtained on the effect of carbon dioxide on various parameters at maturity are presented. No differences were found between plants not exposed to carbon dioxide and plants exposed to CO2 for a 4-week period. This result is not surprising since carbon dioxide treatment was terminated at 46 days after planting and conditions for plant growth were "carbon dioxide limiting”. The trends exhibited by the data presented above indicate a positive response to carbon dioxide in terms of nitrogen fixation in dry beans and that this response is associated with higher net photo— synthetic rates as evidence by the fresh and dry weight of plant parts and by total soluble carbohydrate and organic nitrogen content. Comparisons of the total fresh weight, dry weight, mg soluble carbohy— drates/5 plants and mg organic nitrogen/5 plants clearly indicate this response to carbon dioxide treatment. For example, for the 18-32 dap. carbon dioxide treatment resulted in the following values: 136.58 g fresh weight, 19.3 g dry weight, 443.64 mg of soluble carbohydrate and 636.20 mg of organic nitrogen. In contrast, untreated plants for the same time period totalled: 105.47 g of fresh weight, 18.76 g dry weight, 392.59 mg of soluble carbohydrate and 612.62 mg or organic nitrogen. Similarly, for the 18-46 dap period, untreated plants totalled: 333.30 g fresh weight, 45.35 g dry weight, 1,485.25 mg of soluble carbohydrate and 1,250.36 mg of organic nitrogen. In contrast, plants exposed to carbon dioxide for a period of two weeks 83 Table 11. Effect of carbon dioxide on components and subcomponents of yield of the dry bean variety Porrillo Sintetico at maturity. Treatment1 Igai£_ -C02 (95 d.a.p.) +C02 (18-46 d.a.p.)2 Dry weight of pods 10.88 9.46 Seed yield (W) 8.11 7.30 No. pods (X) 6.75 5.88 No. seeds/pod (Y) 6.19 5.85 Weight of 100 seeds (Z) 19.63 20.33 Stem and root dry weight 3.31 3.95 Node number 11.07 11.05 1 no statistical significance in any of the trait comparisons was obtained. 2 treatment consisted of C02 exposure of plants from 18 to 46 d.a.p. and harvested at 95 d.a.p. 84 (32-46 dap) totalled: 346.48 g fresh weight, 58.06 g dry weight, 1,772.01 mg soluble carbohydrate and 1,635.28 mg of organic nitrogen. For the 4-week carbon dioxide treatment (18.-46 dap) plants totalled: 372.72 g fresh weight, 56.88 g dry weight, 1,862.44 mg soluble carbohydrate and 1,420.39 mg of organic nitrogen. Previously, it had been shown that the concentration of soluble carbohydrate in nodules of plants exposed to carbon dioxide at 18~32 dap was lower than that of untreated plants. An opposite trend has been found for soluble carbohydrate content in nodules for the same treatment comparison. Considering this information with the values obtained for fresh weight of nodules one can suggest that such a response reflects the possibility that carbohydrate is being used at this point for the build-up of nodule mass and that possibly, carbon dioxide treated plants utilize their energy supply at a faster rate to drive the fixation process. The questions posed at the beginning of this section can now be answered. It is suggested that the data presented in this chapter provides a sound basis to propose that carbon dioxide treatment does result in higher levels of photosynthetic products in bean plants. Higher total amounts of soluble carbohydrate in nodules have been found in carbon dioxide treated plants. The concentration of soluble carbohydrates in nodules is lower in carbon dioxide treated than in untreated plants. Higher rates of nitrogen fixation were found in plants exposed to a concentration of carbon dioxide higher than that found in the atmosphere. In contrast to the findings of Hardy and Havelka (1973, 1975) with soybeans, the duration of nitrogen fixation was not extended through carbon dioxide treatment nor was the decline in the rate of nitrogen fixation delayed. 85 REFERENCES Hardy, R. W. F. and U. D. Havelka. 1975. Photosynthate as a major factor limiting N2 fixation by field grown legumes with emphasis on soybeans. In "Symbiotic Nitrogen Fixation in Plants." PA$.Nutman, ed. Cambridge Univ. Press. New York. pp. 421-439. Havelka, U. D. and R. W. F. Hardy. 1975. Legume N2 fixation as a problem of carbon nutrition. In "Proceedings of the lat. International Symposium on Nitrogen Fixation." W. E. Newton and C. J. Nyman, eds. Washington State University Press, Pullman, Wash. Vol. 2. pp. 456—475. Hardy, R. W. F. and U. D. Havelka. 1973. Symbiotic nitrogen fixation: multifold enhancement by COz-enrichment of field grown soybeans. Plant Physiol. 51: 535. Quebedeaux, B. and R. W. F. Hardy. 1975. Reproductive growth and dry matter production of Glycine max (L.) Merr. in res- ponse to oxygen concentration. Plant Physiol. 55: 102-107. Quebedeaux, B, U. D. Havelka, K. L. Livak and R. W. F. Hardy. 1975. Effect of altered p02 in the aerial part of soybean on symbiotic N2 fixation. Plant Physiol. 56: 761-764. Riedels, F. 1922. Kohlensauredungung. Technik. Landw. 3: 87—89. CHAPTER 4 SUMMARY AND CONCLUSIONS The ontogenetic development of four dry bean cultivars has been studied with reference to the relationships that may exist between symbiotic nitrogen fixation and the energy supply (in the form of carbohydrates) to the nodules. Particular emphasis has been placed in these studies on accumulating kinds of information that allow deductions to be made concerning the sequence of events that lead to the observed decline in the fixation of molecular nitrogen in bean plants. The data that previously have been presented are consistent with the hypothesis that carbohydrate supply to the nodules is a limiting factor to the fixation process. It was also shown that an increase of total photosynthate available to the symbiotic system (through CO2 enrichment) results in higher rates of nitrogen fixation. The possibility that nodules and reproductive sinks "compete" for energy has been entertained as a possible explanation for the decline in nitrogen fixation rate in the bean cultivars studied. Spiegelman (1945), has clearly defined the concept of physiological competition: "Every group of biological units (species, individual, cells) are in physiological competition if, a) they require and draw upon a common food source (substrate) and/or, b) excrete harmful metabolites into a common environment". 86 87 The premise given in point "a" has served as a basis for elucida- ting whether competition can be utilized as an explanatory concept of the interacting relationships in a symbiotic system in terms of energy requirements in dry bean plants. The basic assumption that a common substrate supply is available to nodules and reproductive structures is suggested not to be upheld by the responses observed in the cultivars used in this study. The data presented in previous chapters have been interpreted as indi- cating a nutritional dependency upon photosynthate produced in lower leaves by the main nodule population. Events that have been found to be correlated in time with the decline in N2 activity are illustrated in Figure 27. It is suggested that whether a selected pathway is entered rests upon whether or not activating stimuli (genetically and developmentally programmed stimuli) are received: if they are, as may be the case in the decline of N2 fixing activity, senescence of the primary nodule population and lower leaves ensues. It is this author's opionion that a developmentally compensated genetic program predisposes the nodules to the observed decline in N2 fixation. Thus, loss of nodule competency in N2 fixation may not be triggered by a reduction in carbohydrate movement to such structures but by an activating signal(s) affecting both nodule and lower leaf senescence, probably hormonal in nature. This interpretation is also compatible with the results obtained concerning the small burst of nodule activity exhibited subsequent to the decline in N2 fixation, and the increase in carbohydrates in the root system after this decline. The carbohydrate source that accounts for this latter observation is suggested to be different than that due to the lower leaves of the plant, 1. e., a distinct "physiological space and time" is shared by the main nodule 88 population and the lower leaves. These spatial and temporal events are consequences of the unfolding of a genetic program that leads to a dynamic regulation of growth in symbiotic systems. The nature of this regulation remains unknown. Data presented have been inter— preted as supporting the ontogenetic sequence of events outlined by broken lines in Figure 27. Evidence has also been presented concerning the relevance of the symbiotic fixation of molecular nitrogen to legumes. This phenomenon can certainly be considered the insurance of survival of a legume. It has been shown, for example, that nitrogen in the bean plant is stored temporarily in the leaves and it is suggested that mobilization of this nitrogen to the seeds is primarily derived from the ageing processes of bean leaves. A similar phenomenon of mobilization of carbohydrates (temporarily stored in the stems and leaves) can be surmdzed from the data presented in this thesis. 89 «cununuroun uuunsatfl HIIII IIIIIII J sous-«ou/é .suoH no.6.» In rllitllcl II n.1IIIIILHF.IIIIIIIIIIIIIH flouuunarav unavOR ...hntuvv nun menu ”0......” _ .Iul lllllllll '— . _ _ r! cpunmuu v; unconnauu yen ”.----HIIIIL ...nomuuefi snare ... “augu- onuvu ..II. ..uubnuuo shun-sum cm No nuan— ""-l|'-"IIL H-IIIIIIIMHJ “WOCDOV‘ _ van nouquvuxnmnuu_ _ excue o>uuuuuour~u_ PI nu ulnar sou-..."... — eunuOuu M thanonnUV IIIUIIIJ I]... 25:: a“ £33: tour "0.2:? _ _ ovens». Munucmnur w L LIIIJ .Ou-u suntan o’wuquou «on. an occouuuanu .l. IIJIIIIIIIIIL rill! .I. «Hanan HqucoCcouH>cm cauungcaonou HaueotnoHs>or— v uuoHnuLs use “non “ma aunasnausu wean-Haven oucouoouuq _ : n 7-- ---'J . uuunuo n . .....IIIIIII _ a... 833.8 " ovuuunvouron No aunhmouvzn . oHuou consensus: cu. . canvau cauuocuuo sauce». :33? 3.2.0 «on I L....uluquml L noaunuarduua nuuaun] than»? _ Inxus on (sunk-x IJucoHunUhouuuu «use Custom” . we avsbuc uuynromovyv «as. coauoxouuus m» souuanoso Nu unannonou I and graced» nodunuuucoo mason» III-IIIIIH. Ioruoo cannon wad abuunuhou omnuuu . In 0 no oaun. “HHHHHHHHH I.uo use: «uuu4 . PI. u u» 6* nuanuurm 1 «kooonnuu an: nauvnucao . Qua-I was ”Hus canal .eeHu-xuu «k you havens ".04.: GNU _ (our: ouuonngncufiul. ICE g a E 8%“ g on“ 93:5. 10. 11. 90 SELECTED BIBLIOGRAPHY Allen, E. K. and 0. N. Allen. 1958. Biological aspects of symbiotic nitrogen fixation. In "Encyclopedia of Plant Physiology." F. C. Steward, ed. Academic Press, N.Y. 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Poly-B-hydroxybutyrate utilization by soybean (Glycine max Merr.) nodules and assess- ment of its role in maintenance of nitrogenase activity. Plant Physiol. 47: 750-755. Virtanen, A. I. and J. K. Miettineu. 1963. Biological nitrogen fixation. In "Encyclopedia of Plant Physiology." F. C. Steward, ed. 5: 539-668. Yoshida, S., D. A. Forno and J. H. Cock. 1971. Laboratory Manual for Physiological Studies of Rice. The International Rice Research Institute. Los Banos, Phillippines. pp. 61. 94 APPENDIX Least significant differences, henceforth referred to as LSD, refer to the following treatment comparisons: LSDl: LSDZ: difference between difference between difference between difference between 0.01 - probability 0.001 - probability two harvest means. two variety means. two variety means at the same harvest. two harvest means at the same variety. level level 95 Appendix Table 1. Dry weights(1n grams/15 plants) of primary roots of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 1 0.85 0.79 0.84 0.71 0.80 2 1.50 1.39 1.25 1.53 1.42 3 1.91 2.60 2.06 2.48 2.26 4 2.97 4.33 3.64 3.93 3.72 5 4.21 5.07 4.48 6.13 4.97 6 5.74 7.51 6.73 9.32 7.33 7 6.24 9.94 8.83 9.04 8.52 8 6.99 12.14 8.94 10.66 9.68 9 9.40 12.48 10.25 13.07 11.30 10 7.45 12.73 8.93 12.98 10.52 Cultivar Means 4.73 6.90 5.59 6.98 LSD - 1.65; LSD = 0.52; LSD = 1.64; LSD = 2.17 l 2 3 4 S.0.V.1 d.f. M.S. Total 159 Block 3 16.40 Harvest (H) 9 246.07** Error (a) 27 5.20 Cultivar(V) 3 47.36** H X V 27 4.35** Error (b) 90 1.36 Appendix Table 2. 96 Dry weights of "secondary roots" (in grams/15 plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 1 2.66 3.09 1.90 .96 2.65 2 4.71 3.62 4.97 .59 4.97 3 3.88 3.69 3.40 .83 3.70 4 4.91 4.19 3.32 .81 4.06 5 5.32 5.12 5.54 .69 5.42 6 6.51 6.08 4.13 .60 6.08 7 5.99 6.62 4.81 .54 5.99 8 5.87 5.65 5.15 .77 5.61 9 8.04 5.46 5.97 .62 7.02 10 6.30 7.39 5.96 .67 6.83 Cultivar Means 5.42 5.09 4.51 .91 LSDl - 1.90; LSD2 - 0.61; LSD3 a 1.93; LSD4 = 2.52 S.O.V.1 d.f. M.S Total 159 Block 3 8.65 Harvest (H) 9 31.41** Error (a) 27 6.85 Cultivar (V) 3 13.71** H X.V 27 2.17 Error (b) 90 1.88 Appendix Table 3- 72 VUL 26689 S.O.V.1 Total Regression Error SOOOVOI Intercept ICA Pi ao s.o.v.1 Total Regression Error S.0.V.1 Intercept H H2 H3 97 Cubic polynomials fitted to stem dry weight (dependent variable) of four dry bean cultivars, and corresponding Analysis of Variance. d.f. B Values 8.21570833 - 8.31755522 2.56722305 - 0.143.59295 d.f. B Values 4.17288333 - 3.49569464 1.28802054 - 0.06285679 658.35** 14.44 T for Ho: B # 0 1.114 - 1.504 2.251 - 2.100 465.64 2.052** T for Ho: B # 0 1.501 - 1.678 2.997 - 2.439 0.958 Prob T 0.308 0.183 0.065 0.080 0.991 Prob T 0.184 0.144 0.024 0.050 98 Appendix Table 3. (continuation). NEP-Z S.0.V.1 d.f. M.S. R2 Total 9 0.984 Regression 3 318.35** Error 6 2.56 S.O.V.l B Values T for Ho: B f 0 Prob T Intercept 3.59272500 1.156 0.291 H - 3.20814117 - 1.378 0.217 H2 1.29599184 2.698 0.036 H3 - 0.07289350 - 2.531 0.045 Porrillo Sintetico S.O.V.1 d.f. M.S. R2 Total 9 0.988 Regression 3 439.02 Error 6 2.68 S.O.V.1 B Values T for Ho: B i 0 Prob T Intercept 5.66408333 1.781 0.125 H - 5.33321465 - 2.238 0.067 H2 1.84330114 3.750 0.009 3 H - 0.10446528 - 3.544 0.012 I“ by .. I 1 Oui Appendix Table 4. 99 Dry weights of leaves (in grams 15/plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Anaylsis of Variance. Cultivar Harvest 1. Means Harvest l 2 3 l 5.17 5.26 4.82 5.46 5.18 2 8.48 7.33 8.78 10.14 8.68 3 20.40 19.37 17.86 18.32 18.98 4 39.78 40.10 41.31 40.35 40.38 5 52.19 38.48 31.97 48.22 42.72 6 55.20 52.94 47.56 55.21 52.73 7 61.44 57.06 48.06 61.34 56.98 8 48.05 51.45 35.95 47.81 45.82 9 58.55 66.12 51.50 61.96 59.33 10 39.16 53.69 37.10 48.38 44.58 Cultivar Means 38.84 39.18 32.49 39.72 LSD1 - 8.68; LSD2 = 4.52; LSD3 = 14.31; LSD4 = 15.08 S.O.V.1 d.f. M.S Total 159 Block 3 384.31 Harvest (H) 9 6,168.69** Error (a) 27 143.08 Cultivar (V) 3 461.56* H X V 27 75.50 Error (b) 90 103.67 100 Appendix Table 4a. Cubic polynomials fitted to leaf dry weight (dependent variable) of four cultivars, and corresponding Analysis of Variance. 72 VUL 26689 S.O.V.1 d.f. M.S R2 Total 9 0.936 Regression 3 1,194.40** Error 6 40.79 S.0.V.1 B Values T for Ho: B # 0 Prob T Intercept - 7.55600833 - 0.609 0.564 H 7.92070013 0.852 0.427 H2 1.52341856 0.795 0.457 n3 - 0.18452399 — 1.606 0.159 ICA Pijao S.O.V.1 d.f. M.S R2 Total 9 0.945 Regression 3 l,308.50** Error 6 38.25 S.O.V.l B Values T for Ho: B i 0 Prob T Intercept - 5.35578333 - 0.446 0.671 H 6.94349747 0.772 0.470 H2 1.09787879 0.592 0-576 83 - 0.11874747 - 1.067 0.327 101 Appendix Table 4a. (Continuation). NEP-2 S.O.V.1 d.f. M.S. R2 Total 9 0.848 Regression 3 701.89** Error 6 63.09 S.0.V.1 B Values T for Ho: B % 0 Prob T Intercept - 9.16549167 - 0.59458 0.574 H 11.48045770 0.99360 0.359 H2 - 0.14266098 - 0.05986 0.954 H3 - 0.05286869 - 0.36995 0.724 Porrillo Sintetico S.O.V.1 d.f. M.S. R2 Total 9 0.931 Regression 3 1,216.80 Error 6 45.26 S.O.V.1 B Values T for Ho: B # 0 Prob T Intercept - 7.47554167 - 0.572 0.589 H 8.89930250 0.909 0.398 H2 1.00721606 0.499 0.636 3 H - 0.13398038 - 1.107 0.311 Appendix Table 5. 102 Dry weights of vegetative structures (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 l 10.52 11.14 9.04 10.75 10.36 2 17.21 14.60 17.51 21.34 17.66 3 30.55 30.40 27.63 29.24 29.46 4 57.27 58.85 57.60 58.22 57.99 5 78.21 61.33 53.43 76.86 67.46 6 92.56 86.90 79.19 93.82 88.12 7 101.94 99.10 83.73 105.14 97.48 8 91.39 100.97 73.79 93.43 89.89 9 122.91 117.68 99.92 121.49 115.50 10 88.62 110.89 80.69 103.19 95.85 Cultivar Means 69.12 69.19 58.25 71.35 LSD1 - 15.84; LSD2 - 7.02; LSD3 - 22.19; - 24.81 S.O.V.1 d.f. Total 159 Block 3 1,458.26 Harvest (H) 9 21,717.77** Error (a) 27 476.53 Cultivar (V) 3 1,395.98** H X V 27 166.73 Error (b) 90 249.36 Appendix Table 6. 103 Dry weights of flowers and rachis (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 .994 .505 .517 .489 .626 6 2.070 1.042 1.414 .729 1.314 7 2.764 1.268 1.832 1.200 1.766 8 2.531 1.353 2.206 .994 1.771 9 4.792 3.167 3.765 1.853 3.395 10 3.371 3.326 3.474 1.738 2.977 Cultivar Means 2.734 1.777 2.202 1.167 LSD1 - .910; LSD2 - .400; LSD3 . .979; - 1.216 S.O.V.1 d.f. M.S Total 95 Block 3 3.509 Harvest (H) 5 17.152** Error (8) 15 1.458 Cultivar (V) 3 10.797** H X‘V 15 .740 Error (b) 54 .477 104 Appendix Table 7. Dry weight of pods (in grams/15 plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 7 2.823 .653 .622 1.276 1.343 8 11.891 4.552 6.488 2.801 6.433 9 22.885 13.958 18.075 8.124 15.761 10 28.441 27.909 31.305 10.596 24.563 Cultivar Means 16.51 11.768 14.123 5.699 LSD1 - 5.465; LSD2 = 4.612; LSD3 8 9.224; LSD4 = 9.567 S.O.V.l d.f. M.S. Total 63 Block 3 112.850 Harvest (H) 3 1,688.076** Error (a) 9 46.705 Cultivar (V) 3 344.520** H X V 9 78.116 Error (b) 36 41.577 App 105 endix Table 8. Dry weights of reproductive structures (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 .994 .505 .517 .489 .627 6 2.070 1.042 1.414 .729 1.314 7 5.587 1.921 2.455 2.476 3.110 8 14.421 5.905 8.695 3.795 8.204 9 27.677 17.126 21.841 9.977 19.155 10 31.812 31.235 34.779 12.334 27.540 Cultivar Means 13.760 9.622 11.617 4.967 LSD1 a 5.160; LSD - 3.300; LSD3 = 8.083; LSD4 = 8.637: S.O.V.l d.f. M.S Total 95 Block 3 110.85 Harvest (H) 5 1,937.53** Error (a) 15 49.90 Cultivar (V) 3 337.85** H X V 15 80.94 Error (b) 54 32.50 Appendix Table 9. 106 Total dry weights of plants (in grams/15 plants) of ten sequential harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 2 3 4 1 10.52 11.14 9.04 10.75 10.36 2 17.21 14.60 17.51 21.34 17.66 3 30.55 30.40 27.63 29.24 29.46 4 57.27 58.85 57.60 58.22 57.99 5 79.21 61.84 53.95 77.35 68.09 6 94.63 87.94 88.60 94.55 89.43 7 107.52 101.02 86.18 107.62 100.59 8 105.81 106.87 82.48 97.22 98.10 9 150.58 134.80 121.76 131.47 134.66 10 120.44 142.12 155.47 115.52 123.39 Cultivar Means 77.37 74.96 65.22 74.33 LSD1 - 18.61; LSD 8 7.75; LSD3 = 24.51; LSDA = 28.13 S.O.V.1 d.f. M.S. Total 159 Block 3 2,086.48 Harvest (H) 9 30,456.57** Error (a) 27 658.41 Cultivar (V) 3 1,136.39* H X V 27 201.90 Error (b) 90 304.31 *— Appendix Table 10. 107 Nitrogen fixation rates (u moles C2H4 Splalhr‘l) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 2 3 4 l 10.05 9.21 2.89 5.16 6.83 2 31.86 7.97 10.18 23.94 18.49 3 46.51 40.34 13.02 21.63 30.37 4 60.80 46.55 43.96 86.08 59.35 5 81.39 61.05 32.81 57.48 58.18 6 142.08 153.71 108.64 135.32 134.96 7 146.42 96.79 48.14 130.74 105.52 8 19.24 24.72 11.35 23.58 19.72 9 21.50 36.82 41.88 43.60 35.94 10 1.47 13.02 13.98 17.90 11.59 Cultivar Means 56.13 49.02 32.69 54.54 LSD1 - 31.44; LSD = 13.30; LSD3 = 42.08; LSD4 = 48.50 S.O.V.l d.f. M.S Total 159 Block 3 950.10 Harvest (H) 9 28,886.16** Error (a) 27 1,878.68 Cultivar (V) 3 4,591.64** H X V 27 1,090.24 Error (b) 90 896.85 ** ”.2 .01 - probability level *8 | A .05 - probability level Appendix Table 11. 108 Nodule dry weights (in grams/15 plants) of ten sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 1 .4560 .4417 .2014 .2001 .3248 2 .7276 .2870 .2614 .6328 .4772 3 1.4379 .7859 .6567 1.0468 .9818 4 2.5068 2.3003 1.7847 2.9301 2.3805 5 3.7156 2.8615 2.4737 4.3045 3.3388 6 5.8262 5.0052 3.8700 5.8297 5.1328 7 2.8484 5.4224 2.8046 5.3364 4.1029 8 1.6524 2.6344 1.7574 2.6445 2.1722 9 2.6257 3.0136 3.4171 4.0176 3.2685 10 .8156 2.1934 1.3404 3.4278 1.9443 Cultivar Means 2.2612 2.4945 1.8567 3.0370 LSD I 1.1812; LSD = .4274; LSD - 113514; LSD - 1.6562 1 2 3 4 S.O.V.1 d.f. M.S. Total 159 Block 3 1.82 Harvest (H) 9 39.60** Error (a) 27 2.65 Cultivar (V) 3 9.71* H X V 27 1.63 Error (b) 90 .92 Appendix Table 12. 109 Total content of ethanol soluble carbohydrates in nodules (in mg/15 plants) of eight sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance. Cultivar Harvest Means Harvest 1 3 4 3 83.855 45.787 39.447 53.850 55.735 4 124.010 123.09 117.097 157.750 130.487 5 170.477 151.95 147.375 238.835 177.161 6 398.817 361.325 327.422 391.492 369.764 7 150.270 281.625 182.250 298.137 228.071 8 89.300 145.805 94.587 133.827 115.880 9 162.612 199.710 276.907 316.862 239.023 10 40.647 132.575 79.83 177.672 107.681 Cultivar Means 152.499 180.234 158.115 221.053 LSD1 = 97.366; LSD2 = 36.549; LSD3 = 103.375; LSD4= 131.506 S.O.V.l d.f. M.S Total 127 Block 3 13,127.55 Harvest (H) 7 157.748.60** Error (a) 21 17,529.74 Cultivar (V) 3 30,979.48** H X V 21 5,993.84 Error (b) 72 5,380.81 110 Appendix Table 13. Concentration of ethanol soluble carbohydrates in nodules (in mg/g nodule dry wt.) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 3 62.85 60.97 58.18 55.93 59.48 4 54 03 50 95 60.78 52 47 54 56 5 45.92 50.92 60.16 54.67 52.92 6 65.83 73.50 60.07 62.23 66.16 7 52.51 51.68 61.37 51.91 54.37 8 52 81 54 90 53.95 51 28 53 24 9 61.65 69.82 80.22 78.07 72.44 10 49.00 60.72 62.00 55.85 56.89 Cultivar Means 55.58 59.18 62.09 58.18 S.0.V.1 d.f. M.S. Total 127 Block 3 345.12 Harvest (H) 7 794.40 Error (a) 21 355.72 Cultivar (V) 3 232.08 H X V 21 95.90 Error (b) 72 121.47 111 Appendix Table 14. Total starch content in nodules (in mg/lS plants) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 3 131.580 80.880 67.747 141.795 105.501 4 406.220 369.402 247.982 430.502 363.527 5 609.610 435.530 342.777 835.820 555.934 6 780.745 773.420 520.505 775.200 712.467 7 315.607 585.837 321.362 427.792 412.650 8 164.370 284.305 147.667 217.030 203.343 9 285.990 259.565 317.177 363.527 306.565 10 54.040 207.427 103.265 239.615 151.088 Cultivar Means 343.520 374.546 258.561 428.910 LSD1 - 161.892; LSD2 - 87.016; LSD3 - 246.120; LSD4 = 266.394 S.O.V.1 d.f. M.S. Total 159 Block 3 98,968.66 Harvest (H) 7 687.144.09** Error (a) 21 48,463.50 Cultivar (V) 3 162,398.09** H X V 21 34,338.43 Error (b) 72 30,500.59 Appendix Table 15. 112 Concentration of starch in nodules (mg/g dry weight) of eight sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 3 90.94 80.58 83.12 99.58 88.56 4 144.87 163.94 138.89 144.87 148.14 5 163.97 163.65 152.17 198.12 169.48 6 129.82 150.35 133.45 143.27 139.22 7 109.97 107.60 113.12 82.38 103.27 8 100.95 104.55 82.30 83.52 92.83 9 105.87 88.37 85.27 84.05 90.89 10 65.77 88.15 80.25 78.10 78.07 Cultivar Means 114.02 118.40 108.57 114.24 LSD1 8 26.358; LSD4 = 46.521 S.O.V.1 d.f. M.S Total 127 Block 3 6,451.91 Harvest (H) 7 18,092.97** Error (a) 21 1,284.65 Cultivar (V) 3 519.64 H X V 21 609.16 Error (b) 72 998.83 Appendix Table 16. 113 Total ethanol soluble carbohydrate content of primary roots (in mg/15 plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 21.645 25.542 22.81 28.510 24.627 6 43.252 54.605 46.045 61.437 51.335 7 37.415 55.087 61.527 51.182 51.303 8 55.797 87.362 70.040 80.530 73.432 9 71.730 106.435 91.067 119.337 97.142 10 61.827 108.137 79.562 127.965 94.373 Cultivar Means 48.611 72.862 61.842 78.160 LSDl I 19.304; LSD2 = 8.372; LSD3 = 20.506; LSD4 = 26.023 S.O.V.l d.f. M.S Total 95 Block 3 2,458.31 Harvest (H) 5 12,705.72** Error (a) 15 656.46 Cultivar (V) 3 43104-17** H X V 15 504.75 Error (b) 54 209.21 114 Appendix Table 17. Concentration of ethanol-soluble carbohydrates of primary roots (mg/g dry weight) of six sequential weekly harvests of four dry bean. cultivars,and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 5 5.056 4.892 5.027 4.794 4.942 6 7.509 7.272 6.746 6.618 7.036 7 6.017 5.601 6.906 5.575 6.032 8 7.851 7.266 7.527 7.496 7.535 9 7.751 8.504 8.830 9.145 8.558 10 8.511 8.478 8.943 9.835 8.942 Cultivar Means 7.117 7.007 7.330 7.244 LSD1 - 1.291; LSD4 = 1.831 S.O.V.l d.f. M.S. Total 95 Block 3 .557 Harvest (H) 5 36.721** Error (a) 15 2.937 Cultivar (V) 3 .483 H X V 15 1.012 Error (b) 54 1.156 Appendix Table 18. 115 Total starch content of primary roots (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 5 41.890 52.857 46.870 53.057 48.669 6 73.377 69.125 69.010 124.445 83.989 7 104.500 152.260 138.910 124.585 130.064 8 200.665 344.327 269.062 314.822 282.219 9 254.325 297.097 264.550 393.540 302.378 10 244.870 348.625 265.097 472.260 332.713 Cultivar Means 153.271 210.715 175.583 247.118 LSDl - 66.845; LSD2 = 33.202; LSD3 = 81.327; LSD4 = 96.356 S.O.V.l d.f. M.S Total 95 Block 3 20,512.52 Harvest (H) 5 243,322.63** Error (a) 15 7,871.55 Cultivar (V) 3 40,563.26** H X V 15 7,720.12 Error (b) 54 3,290.57 116 Appendix Table 19. Concentration of starch of primary roots (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 9.824 10.993 10.251 8.537 9.901 6 12.722 9.572 9.941 13.214 11.362 7 16.338 15.401 15.416 13.652 15.202 8 28.525 28.228 29.926 29.652 29.083 9 26.341 23.541 25.269 29.738 26.222 10 32.628 27.224 30.481 36.391 31.681 Cultivar Means 21.063 19.160 20.214 21.864 LSD1 - 2.055; LSD4 - 5.733 S.O.V.1 d.f. M.S Total 95 Block 3 32.328 Harvest (H) 5 1,456.921** Error (a) 15 14.884 Cultivar (V) 3 32.261 H X V 15 16.054 Error (b) 54 16.431 Appendix Table 20. 117 Total ethanol soluble carbohydrate content of "secondary roots" (in mg/15 plants) of six sequential weekly harvests of tour dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 110.005 141.417 173.127 125.285 137.459 6 237.080 243.640 202.695 287.117 242.633 7 300.802 328.015 240.295 249.047 279.540 8 292.722 301.932 254.297 244.877 273.457 9 434.830 341.020 372.930 539.662 422.111 10 225.957 305.257 171.305 347.527 262.512 Cultivar Means 266.900 276.880 235.775 298.920 LSD1 . 65.639; LSD2 = 45.819; LSD3 8 112.233; LSDA - 116.593 S.O.V.l d.f. M.S Total 95 Block 3 28,672.07 Harvest (H) 5 133,158.20** Error (a) 15 7,590.01 Cultivar (V) 3 16,512.51 H X V 15 11,395.57 Error (b) 54 6,266.79 Appendix Table 21. 118 Concentration of ethanol-soluble carbohydrates of "secondary roots" (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 3 5 21.18 26.91 32.06 21.38 25.38 6 37.15 40.62 47.97 39.91 40.66 7 49.45 49.64 50.28 38.49 46.96 8 50.27 55.93 50.11 44.16 50.12 9 56.67 64.30 61.58 61.97 61.13 10 36.62 40.82 28.56 45.10 37.77 Cultivar Means 41.89 46.37 45.09 41.34 LSD1 - 8.63; LSDA - 12,837 S.0.V.1 d.f. M.S Total 95 Block 3 $18.39* Harvest (H) 5 2,353.50** Error (3) 15 131.32 Cultivar (V) 3 143.29 H X V 15 106.21 Error (b) 54 61.52 Appendix Table 22. 119 Total content of starch of "secondary roots" (in mg/15 plants) of six sequential weekly harvests of four dry bean cultivars, and corres- ponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 5 224.297 258.965 218.370 271.057 243.172 6 319.977 316.952 194.457 332.605 290.998 7 363.605 366.880 227.03 319.982 319.374 8 402.840 384.037 401.54 377.215 391.408 9 717.282 518.135 481.265 1,137.427 713.527 10 492.125 588.175 396.817 1,054.515 632.908 Cultivar Means 420.021 405.524 319.914 582.134 LSD1 - 121.781; LSD2 - 120.259; LSD3 = 294.573; LSD4 = 281.611 S.0.V.1 d.f. M.S Total 95 Block 3 64,642.20 Harvest (H) 5 606,372.10** Error (a) 15 26,126.57 Cultivar (V) 3 287.583.11** H X V 15 90,465.96 Error (b) 54 43,170.48 120 Appendix Table 23. Concentration of starch of "secondary roots" (mg/g dry weight) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest l 2 3 4 5 41.47 50.45 42.32 48.20 45.61 6 50.95 52.76 46.09 43.07 48.22 7 60.64 55.06 47.96 51.08 53.69 8 70.14 72.08 77.56 64.60 71.09 9 63.24 94.01 81.39 43.10 70.44 10 76.19 78.73 66.96 82.04 75.98 Cultivar Means 60.44 67.18 60.38 55.35 LSD1 - 17.66; LSD4 - 32.39 S.0.V.1 d.f. I M.S. Total 95 Block 3 419.92 Harvest (H) 5 2,780.24** Error (a) 15 549.73 Cultivar (V) 3 566.19 H X V 15 391.74 Error (b) 54 477.98 121 Appendix Table 24. Total content of ethanol soluble carbohydrates of stems (in mg/15 plants) of six sequential weekly harvests, of four dry bean cultivars. and corres- ponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 1,479.62 1,164.62 945.70 1,383.00 1,243.24 6 2,503.10 1 832.40 2,757.72 2,082.90 2,294.03 7 4,427.97 2,081.80 3,289.87 2,852.37 3,163.01 8 5,095.32 3,500.60 3,591.27 4,204.77 4,097.99 9 4,911.45 2,778.70 3,218.27 2,641.25 3,387.42 10 5,432.42 4,163.30 4,159.15 3,722.75 4,369.41 Cultivar Means 3,974.98 2,586.90 2,993.67 2,814.51 LSD1 = 1,240.06: LSD2 = 655.52; LSD3 = 1,605.93; LSD4 = 1,848.86 S.O.V.1 d.f. M.S Total 95 Block 3 11,740,198.8 Harvest (H) 5 21,730,494.7** Error (a) 15 2,708,964.9 Cultivar (V) 3 8,971,611.0** H X V 15 899,391.3 Error (b) 54 1,283.093.l Appendix Table 25. 122 Concentration of ethanol soluble carbohydrates of stems (mg/15 plants) of six sequential weekly harvests of four dry bean cultivars, and corres— ponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 111.18 108.87 104.74 107.51 108.83 6 127.28 127.43 165.39 130.41 137.63 7 180.10 105.86 165.60 110.40 140.49 8 175.19 121.98 153.49 156.22 151.72 9 109.37 87.30 114.71 77.10 97.12 10 158.54 117.56 152.79 122.91 137.95 V 143.61 111.50 143.29 117.43 LSD1 B 42.01; LSD2 = 20.18; LSD3 = 49.42; LSD4 = 59.51 S.O.V.1 d.f. M.S. Total 95 Block 3 2,008.85 Harvest (H) 5 7,123.23 Error (a) 15 3,109.54 Cultivar (V) 3 6,861.36** H X V 15 1,040.26 Error (b) 54 1,215.24 123 Appendix Table 26. Total content of starch of stems (in mg/lS plants) of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 1,979.66 662.03 834.22 1,212.23 1,172.04 6 1,837.83 1,293.63 1,449.63 1,639.21 1,555.08 7 4,839.71 2,232.29 2,237.88 2.185.46 2,873.84 8 12,394.75 5,605.19 4,232.45 6,863.23 7,273.91 9 15,289.49 6,947.05 7,413.88 9,937.78 9,897.05 10 7,280.97 5,815.17 4,924.71 6,414.67 6,108.88 Cultivar Means 7,270.40 3,759.23 3,515.46 4,708.77 LSD1 8 3,053.25; LSD2 = 1,020.01; LSD3 = 2,498.50; LSD4 = 3,713.03 S.0.V.1 d.f. M.S Total 95 Block 3 14,817,867.0 Harvest (H) 5 195,884,735.0** Error (a) 15 16,422,812.0 Cultivar (V) 3 70,749,829.0** H X V 15 10,298.160.0** Error (b) 54 3,105,707.0 124 Appendix Table 27. Concentration of starch in stems (mg/g dry weight of six sequential weekly harvests of four dry bean cultivars, and corresponding Analysis of Variance. Cultivar Harvest Means Harvest 1 2 3 4 5 150.06 67.67 99.90 94.75 103.09 6 98.89 99.60 92.10 105.77 99.09 7 200.80 105.91 116.63 90.05 124.35 8 430.98 187.61 199.11 239.66 264.34 9 344.99 225.75 258.84 278.90 275.62 10 213.59 174.61 177.73 207.26 191.94 Cultivar Means 241.03 143.52 156.39 169.40 LSD1 - 64.10; LSD2 - 33.72; LSD3 - 82.61; LSD4 = 95.31 S.O.V.1 d.f. M.S. Total 942 Block 3 1,758.51 Harvest (H) 5 100,681.00** Error (a) 15 7,238.23 Cultivar (V) 3 44,260.14** H X V 15 6,729.78* Error (b) 532 3,402.67 2 - correction l d.f. due to missing observation. Appendix Table 28. 125 Total content of ethanol soluble carbohydrates of leaves (in mg/lS p1ants