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Title of Thesis

Jehan Bakht
Institute/University/Department Details
Department of Biological Sciences/ Quaid-i-Azam University, Islamabad
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
abscisic acid, aba, chickpea, cicer arietinum l, cold tolerance, phytohormones, polypeptides, plasma membrane structure, cold hardiness, chick pea plants

This research project investigated the physiological, biochemical and molecular basis of cold tolerance in chick pea (Cicer arietinum L.) plants. The project was divided in to two parts. During the first part field experiments were conducted at three locations with different climates (Swat I , Swat2 and Islamabad, Pakistan) to evaluate the effect of low temperature and different phytohormones (ABA, IAA and BA) application on various growth parameters (shoot fresh weight, shoot dry weight, shoot length, root fresh weight, root dry weight, root length, number of pods per plant, number of seeds per pod and 1000 grain weight). The results suggest that low temperature had a signifIcant (p < 0.05) detrimental effect on all the growth parameters tested. In addition, foliar spray of ABA (10-4 M) had a profound effect and provided some protection against cold-induced inhibition of growth.

During the second part of the project, chick pea plants were grown in controlled environment growth chambers and exposed to non-freezing (typical of Islamabad) or freezing temperatures (typical of Swat2), with or without exogenous ABA application. A range of growth parameters were measured from these plants and the results were found to be broadly similar to those from the field trial experiments. Furthermore, the growth chamber experiments were used to investigate the biochemical and molecular mechanisms of cold tolerance in chick pea plants. It was evident from the growth chamber experiments that low temperature significantly (p < 0.05) reduced all the growth parameters (shoot fresh weight, shoot dry weight, shoot length, root fresh weight, root dry weight and root length) under study. It was also clear from these experiments that foliar application of ABA partly prevented the adverse effect of low temperature on growth. From these experiments it was concluded that the results observed from field trials could be attributed to the effects of temperature only, and an interaction between temperature and ABA.

Experiments were carried out to study the effect of temperature and exogenous ABA application on gene expression in chick pea plants. Protein was isolated from the treated and control plants and separated by one and two-dimensional SDS-PAGE. These experiments revealed that cold acclimation was accompanied by changes in protein composition and that to some extent, ABA could induce a similar pattern of changes in plants exposed to freezing temperatures. Low temperature and ABA also induce the synthesis of some heat stable proteins, who's function in vivo is unknown. The majority of these ABA and low temperature-induced polypeptides had neutral or basic pI.

Exposure of plants to low temperatures or ABA application both resulted in elevated levels of endogenous ABA which presumably is involved in cell signalling process. In addition, endogenous levels of proline also increased with endogenous ABA levels, although it is doubtful whether this plays a significant role in the acclimation processes reported here.

Experiments were also conducted to study the structure and functions of plasma membrane isolated from control and ABA or cold acclimated plants. Exposure of chick pea plants to low temperature and ABA treatment over a period of 14 days significantly (p < 0.05) decreased the LT so, the temperature at which 50% of cells lysed « -10 QC) when compared with the plants maintained at control temperatures, although at the growth temperatures (> -2°C) no differences were observed.. Furthermore, low temperature exposure increased the proportion of unsaturated fatty acids suggesting changes in the viscosity of the plasma membrane at low temperatures. However, low temperature exposure and ABA treatment did not significantly (p < 0.05) affected membrane viscosity over the 14 days period. Exposure of chick pea plants to low temperature and ABA application did not affect the plasma membrane ATPase activity when compared with plants kept at control temperatures.

It is concluded that both low temperature and ABA treatment results in changes in a number of factors that are considered to be important in cold acclimation (changes in plasma membrane lipid saturation, LT so, protein composition, endogenous ABA levels, endogenous proline levels etc.). These factors may be important in providing protection from frost damage at low, potentially lethal temperatures. However, in this study, plants were not exposed to lethal temperatures (> -6°C) and it is inferred that ABA may have a role in conferring a hither-to un-observed acclimation process which provides chick pea plants with better growth in cool non-lethal temperatures.

Download Full Thesis
4752.31 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents 0
133.98 KB
2 1 Introduction 1
573.51 KB
  1.1 Chick Pea 1
  1.2 Mechanism Of Frost Damage To Plants 3
  1.3 Adaptation To Frost Damage , 6
  1.4 Identification Of Putative Frost Tolerant Genes , 13
  1.5 Expression Of Cold Regulated Genes In Response To ABA 15
  1.6 Expression Of Cold Regulated Genes By Drought Stress , 17
  1.7 Cellular Location Of Cold Regulated Polypeptides , 17
  1.8 Conservation Of Cold Regulated Genes , 18
  1.9 Abscisic Acid , 18
  1.10 Lea Proteins , 25
  1.11 Aims And Objectives Of The Project , 27
3 2 Materials And Methods 29
584.28 KB
  2.1 Field Experiments , 29
  2.2 Laboratory Based Experiments , 34
4 3 Growth Parameters 54
931.32 KB
  3.1 Introduction , 54
  3.2 Field Experiments , 55
  3.3 Growth Room Experiments , 60
  3.4 Ultrastructure Of Leaf Tissue , 66
  3.5 Summary , 67
5 4 Gene Expression 68
880.1 KB
  4.1 Introduction , 68
  4.2 One-Dimensional SDS-PAGE Of Polypeptides , 70
  4.3 Two-Dimensional SDS-PAGE 77
  4.4 Summary 80
6 5 Endogenous ABA And Proline Levels 83
575.84 KB
  5.1 Introduction 83
  5.2 Endogenous Levels Of ABA 85
  5.3 Endogenous Proline Levels 87
  5.4 Summary 89
7 6 Plasma Membrane Structure 91
553.04 KB
  6.1 Introduction 91
  6.2 Assessment Of Cold Hardiness (LT 50 ) 92
  6.3 Fatty Acid Composition And Cold Tolerance 95
  6.4 Fluorescence Polarisation 98
  6.5 A Tpase Activity 103
  6.6 Summary 104
8 7 Discussion 106
202.52 KB
  7.1 Effect Of Low Temperature And ABA On Physiology Of Chick Pea Plants 106
  7.2 Gene Expression 109
  7.3 Endogenous ABA And Proline Concentration 113
  7.4 Plasma Membrane Structure And Function 116
  7.5 Conclusions 119
9 8 References 120
301.34 KB
10 9 Appendix 121
317.63 KB