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Preface Plant Physiology is the basic science which caters to the need of several cognate disciplines such as Genetics & Plant Breeding, Horticulture, Agronomy, Environmental Sciences, Soil Science, Plant Molecular Biology etc. Several of the physiological tools and traits are commonly used in research in field of all cognate disciplines in improving crop productivity. For example, the growth analysis although is very old concept but provides the basis of a plant species or a variety to perform better or poor and thus elucidates the causes which a breeder would like to know and design the future research programme accordingly. Mineral nutrients are the most important components of plant growth and yield. Soil Scientist, Agronomist and Horticulturist are equally concerned about the uptake and utilization of these macro and micro-nutrients by different varieties of the crop. The status of the nutrient could be analyzed in the plant or soil to find the level of the nutrients using modern techniques like Atomic Absorption Spectrophotometer. Photosynthesis is the area of modern science where an effort is being made to either compare the varieties to know the efficiency or to enhance the photosynthetic efficiency, thus measurement of photosynthesis in normal, under abiotic stress environment or biotic stress environment is very relevant and important. Equally important is the carbohydrate, protein and oil content estimation in different plant parts for quantitative and qualitative analysis of the plant material which are useful in our plant breeding research. In the recent past much emphasis has been given to the oxidative stress due to production of free radicals. In almost all research on biotic and abiotic stress, information on the level of oxidative stress and the role of antioxidative enzymes have become very important. In Post Harvest Physiology, efforts are being made to enhance the shelf life of fruits, vegetables and flowers. It is thus very much pertinent to assay the process involved in ripening. Efforts have been made to include common ripening enzymes, the plant pigments and other related parameters to study the ripening process. Alongwith these, some modern techniques of estimation of proteins, nucleic acids have also been given. Plant hormones are very important in regulating the plant growth and development. Most of the commonly involved plant hormones like IAA, GA, ABA and ethylene have been included and the methods of their quantification have been described. Besides, commonly used buffers have also been given which are used in assay of different enzymes. In the end, some tables of important characters and units have also been given for convenience of the readers as ready reference. The whole manual has been given chapterwise/ themewise and the methods for estimation has been described in a very simple way. All efforts have been made to provide the references also so that users can refer book and original articles for more information. It is hoped that this book will be useful to students of Plant Sciences, the research scientists and others who may be interested in physiological analysis in their research work. If this meets the requirement of even few interested scientists/students, the purpose of this book is solved.

1. Chlorophylls Chlorophyll can be regarded as a true representation of health of green plants. This presumption is based on the fact that chlorophyll is most commonly and immensely influenced by biotic and abiotic stress conditions in addition to events like pathogenic invasion, pollution, mineral deficiencies as well as mineral toxicity. This is the reason that estimation of chlorophyll is considered to be one of the most important parameters in plant physiological studies. Eukaryotic plant cells possess many types of ‘reserves’, one of them is plastid. A plastid, that contains green colour is known as chloroplast and the green colouration of a chloroplast is mainly due to the presence of a pigment to which we call as chlorophyll. Normally, chlorophyll constitutes about 4% of chloroplast dry weight. Chlorophyll ‘a’ and ‘b’ are the most abundantly found in all autotrophic higher plants. However, there are other chlorophylls also such as ‘c’, ‘d’, ‘e’, bacteriochlorophyll and bacterioviridin which have restricted presence in blue-green, brown and red algae. A chlorophyll molecule contains ‘Tetrapyrrole’ skeleton formed into a ring with an atom of magnesium (Mg++) in the centre. A so-called ‘Pyrrole’ molecule contains four carbon and one nitrogen atoms arranged in a ring. Four such pyrrols arranged in a ring form the ‘Head’ of chlorophyll molecule. Attached to this is ‘porphyrin ring’, at one point, is a ‘phytol’ tail- a long chain of linked carbon.

The weight of living plant material contained above and below a unit of ground surface area at a given point of time, is known as plant “Biomass”. Biomass is of utter importance because it provides energy source (Roberts et al. 1987). Biomass, which has dimensions of mass per unit, is expressed as g m-2, kg ha-1 or t ha-1. Measurement of Biomass The area (in the crop field) under study is divided into a number of equal squares or rectangular plots measuring 1.0 m2 or 0.5 m2. Each square is designated with a particular number. Sample can be harvested from a small area or quadrate at the centre of each square; the remaining peripheral area serving as buffer zone (Wiegert, 1962). Depending upon the growth pattern of plant species two different designs have been adopted for the sampling:

Growth represents overall performance of a plant under a particular environment. Generally, we restrict to the above ground plant parts for growth studies, however, a better understanding of plant growth and development would be achieved if total dry matter (including above ground shoot as well as underground root) were taken into consideration. The basic principles behind growth analysis constitutes (i) data on plant height and (ii) leaf area. The quantitative analysis of growth is based upon several terms, which are described below (Hunt, 1978). 1. Growth Growth is an irreversible increase in size which may or may not be accompanied by an increase in dry weight. It is the sum total of all the metabolic activities and normally results from the excess of anabolism over catabolism. In general, it refers to quantitative changes in the life cycle of plants. However, there are a few examples where growth is not accompanied by an increase in dry weight such as (i) germinating seeds and, (ii) etiolated plants.

1. Relative Water Content Relative water content (RWC) is an important parameter to measure the water status of plant tissue. High RWC, under moisture stress, denotes ability of plants to tolerate drought. (Ritchi et al. 1990). RWC and water potential have often been reported as indicators of drought tolerance (Schonfeld et al. 1988). Leaf water status may be estimated by measuring the RWC following Barrs and Weatherlay’s (1962) method as given below:

The most abundantly and commonly available compounds in the plant kingdom are carbohydrates. They comprise saccharides viz. Monosaccharides - glucose, fructose etc. Disaccharides - sucrose, lactose, maltose etc.; and Polysaccharides - starch, cellulose etc. Cellulose is the major component of plant cell wall. The active groups in carbohydrates are aldehyde (-CHO) and ketone (=CO). In addition, they contain hydroxyl groups as well. The chemical properties of saccharides vary depending upon the number of hydroxyl groups and the presence or absence of (–CHO) or (=CO) groups.

Plants take up nitrogen from soil either in the form of nitrate or ammonia. Nitrate taken by plants is reduced by nitrate reductase and nitrite reductase enzymes. They catalyze step-wise reduction of nitrate to nitrite and nitrite to ammonia. Ammonia occupies the key position in nitrogen metabolism for the synthesis of organic nitrogen. Glutamic acid dehydrogenase, GOGAT and GS are important enzymes which are involved in the conversion of ammonia into glutamic acid, a primary amino acid. The ?-amino group of several amino acids is formed as a result of transamination reaction through transaminase/ aminotransferase enzymes. This process is of great significance in amino acid metabolism.

1. Protein Estimation The method developed by Lowry et al. (1951) is largely followed for the estimation of soluble protein. This method is quite sensitive and gives a moderately constant value. Protein may also be estimated by analyzing the total nitrogen content. Multiplying total nitrogen value with 6.25 will give the crude protein content. However, the nitrogen per cent varies with amino acid composition of proteins. For more refined expression of protein percentage, different factors are used for different samples. Some of the conversion factors for cereal and oilseed are given below:

1. Deoxyribo Nucleic Acid (DNA) DNA is a genetic material, originally confined to nucleus of a cell. In plants, some DNAs are also present in mitochondria and chloroplasts. DNAs are vital macro molecules having high molecular weight. Isolation of plant DNA is cumbersome or difficult primarily because of the presence of a number of secondary plant products. Secondly, a protocol, which is found suitable for isolation of DNA in one plant, may not work in other. The efficiency of DNA extraction depends on many factors, such as sample material, ionic conditions of the extraction medium and type of lysing agent used etc. There are two methods, one developed by Dellaporta et al. (1983) and the other by Murray and Thompson (1980) which are most commonly used for extraction of DNA in a number of plant genera. Murray and Thompson’s (1980) method is described here below with minor modifications.

Fatty acid esters of glycerol are known as fats or lipids. A fat, in its liquid state, is called ‘oil’. Oilseed plants such as castor, groundnut, sunflower, safflower, linseed etc. contain oil as reserve food in their seeds. It must be noted that a small quantity of free fatty acid is usually present in oils along with the triglycerides. It increases during storage. The free fatty acid content of an oil is called as ‘acid number’ or ‘acid value’. The keeping quality of oil relies upon the free fatty acid content. During germination of oilseeds, lipases play an important role in hydrolyzing the stored oils to fatty acid and glycerol. Fatty acid is metabolized to acetyl-COA, which may take part in glyoxylate cycle forming sugars from fatty acid involving the enzymes- Isocitritase and Malate Synthetase.

Photosynthesis is a very important parameter in plant physiological studies. It is the only process in nature which utilizes solar energy and converts it into chemical energy. It has a direct bearing on plant productivity. By understanding the process of photosynthesis, manipulation of productivity can be made possible. 1. Measurement of Photosynthesis Rate Measurement of CO2 uptake provides an alternate and direct method of measuring productivity, with important advantages over measurements of change in dry weight. It is instantaneous and non-destructive. Infra Red Gas Analyzer (IRGA) is an equipment which is most widely used for the measurement of photosynthesis in situ in the field or in detached leaves under laboratory conditions. It is less time consuming and reliable. It can also be used for the measurement of rate of respiration and CO2 compensation concentration (CO2 CC), provided a little alteration is made in the chamber.

Respiration rate, irrespective of plant parts (viz. leaves, fruits or seeds), is considered to be an important parameter in plant physiological studies. Respiratory gas exchange is just reverse or opposite to that of photosynthesis and as the later is an energy building phenomenon where ATP is stored in the photosynthates produced; respiration involves breakdown of photosynthates for release of energy to be utilized for the performance of other activities of life. Respiration plays an important role during seed germination. A germinating seed requires a lot of energy which is derived from the breakdown of different types of food reserved in the cotyledons. On the basis of respiration a climacteric fruit can well be distinguished from a non climacteric one; as the former reveals a characteristic ‘Climacteric Burst’ in respiration, which is completely lacking in the later. There are a few instruments used for the measurement of respiration in plants viz.:

Free radicals, such as singlet oxygen (1O2), superoxide (O2 .–) and some active derivatives of oxygen, like H2O2 are although generated in plant cells during the course of normal metabolic processes, but their production is notably enhanced when the plants are exposed to any kind of stress environment. The reactive oxygen species damage important cellular components, including cell membrane by peroxidation of lipid and de-esterification of fatty acids. Plants have evolved a wide range of mechanisms to contend with this problem. The antioxidant defence system of a plant comprises of a variety of antioxidant molecules and enzymes which take care of free radicals produced in the cells. These enzymes are popularly termed as the ‘Scavenging Enzymes’ or ‘Antioxidative Enzymes’. Conversion of oxygen molecules into its reactive form leads to induction of a sort of stress in plants, to which we call as ‘Oxidative Stress’. It is the equilibrium between oxidative stress and antioxidant enzymes, which determines the fate of a plant. Plants possess scavenging enzyme system to protect them from destructive oxidative reactions. These enzymes are not restricted to the intercellular compartment but are also found in the apoplast to a limited extent. Some of the enzymes are as follows:-

There is loosening of cell wall of climacteric fruits during ripening. The enzyme pectin methyl esterase (PME), is responsible for the de-esterification of pectin (major constituent of cell wall) into pectic acid (or Polygalacturonic acid) and methanol. Polygalacturonic acid serves as a substrate for PG (Polygalacturonase) enzyme. This enzyme (PG) starts depolymerization of pectic acid leading to the formation of small chains of tri, di and monogalacturonic acids. A large number of pathogens also have been reported to produce pectic enzymes to macerate the plant tissue. Hence, in addition to its role in ripening process and shelf life of fruits, the enzyme PG is also studied in relation to the diseases of the plants.

Plant hormones and growth regulators are compounds that influence flowering, aging, root growth, prevention or promotion of stem elongation, prevention of leaf emergence and/or leaf fall and many other physiological processes. These substances produce major growth changes at a very low concentration. Hormones are endogenously produced substances in plants while plant growth regulators may be synthetic compounds (i.e. IBA and Cycocel) that mimic naturally occurring plant hormones or they may be natural hormones that are extracted from plant tissue (e.g. IAA). These growth regulating substances, most often, are applied as a spray to foliage or as a liquid drench to soil around a plant’s base. Generally, their effects are short-lived and they may need to be re-applied in order to achieve the desired effect. Plant growth regulating substances are normally classified into five groups viz.: (i) Auxins, (ii) Gibberellins; (iii) Cytokinins; (iv) Ethylene; and (v) Abscisic acid. For the most part, each group contains both naturally occurring hormones as well as synthetic substances. Physiological functions of each and every group of growth regulating substances are given below in brief:

Ascorbic acid (vitamin C) is present in almost all fresh fruits and vegetables in varying quantities ranging from 0.02 to 1.0 mg per g fresh weight. It is most abundantly found in bittergourd and berries. The method for estimation of ascorbic acid is given here below as described by Albrecht (1993). It is an easy and simple method based on titration technique.

Phenols constitute a class of aromatic organic compounds having at least one hydroxyl group attached directly to the benzene ring.

A Flame Photometer has long been in use for analysis of elements; however, it is now being gradually replaced by Atomic Absorption Spectrophotometer. The reason being, that the former can analyze only a few elements such as sodium (Na), potassium (K), calcium (Ca), barium (Ba) and lithium (Li), whereas the later can be used to analyze almost all the elements known, provided a specific Hollow Cathode Lamp (HC), for a specific element, is available. In addition, Atomic Absorption Spectrophotometer is more precise and accurate in measurement as compared to Flame Photometer. In this chapter, we shall deal with the operation techniques, working principles and a brief description of each and every component/constituent unit of a Flame Photometer as well as an Atomic Absorption Spectrophotometer.

1. Mitochondria Mitochondria are known as the “Power House” of a cell because they are the site of ATP production. In addition, they are also involved with photorespiration and oxidation of fatty acids and work in association with peroxisomes, glyoxysomes and chloroplasts. Isolation of intact mitochondria is rather difficult. This is because the cell walls of plant tissue are rigid as compared to the single-layered wall surrounding the mitochondria. A significant fraction of mitochondria suffer structural damage during the process of tissue homogenization. Therefore, the pelleted mitochondria need further purification by sucrose gradient centrifugation. Mitochondria can be isolated from green as well as etiolated leaf; however, the latter is preferred to obtain higher yield of mitochondria (Moore and Proudlove, 1983).

In this chapter, we describe the methods of preparation of some of the buffers, most commonly used in the assay of enzymes required for plant physiological and histochemical studies. The buffers have been arranged in an alphabetical order starting with their names. These methods are not necessarily identical with those of the original authors. It is advised that the users should re-determine the titration curves of majority of the buffers. These buffers have also been listed in Method in Enzymology, Vol. 1 (1955), pp. 138-146.

Appendix Table 1: The International system of units