12.6.2 Biological Nitrogen Fixation

Very few living organisms can utilise the nitrogen in the form N₂, available abundantly in the air. Only certain prokaryotic species are capable of fixing nitrogen. Reduction of nitrogen to ammonia by living organisms is called biological nitrogen fixation. The enzyme, nitrogenase which is capable of nitrogen reduction is present exclusively in prokaryotes. Such microbes are called N^2- fixers.

The nitrogen-fixing microbes could be free-living or symbiotic. Examples of free-living nitrogen-fixing aerobic microbes are Azotobacter and Beijerinckia while Rhodospirillum is anaerobic and free-living. In addition, a number of cyanobacteria such as Anabaena and Nostoc are also free-living nitrogen-fixers.

Symbiotic biological nitrogen fixation

Several types of symbiotic biological nitrogen fixing associations are known. The most prominent among them is the legume-bacteria relationship. Species of rod-shaped Rhizobium has such relationship with the roots of several legumes such as alfalfa, sweet clover, sweet pea, lentils, garden pea, broad bean, clover beans, etc. The most common association on roots is as nodules. These nodules are small outgrowths on the roots. The microbe, Frankia, also produces nitrogen-fixing nodules on the roots of non-leguminous plants (e.g., Alnus). Both Rhizobium and Frankia are free-living in soil, but as symbionts, can fix atmospheric nitrogen.

Uproot any one plant of a common pulse, just before flowering. You will see near-spherical outgrowths on the roots. These are nodules. If you cut through them you will notice that the central portion is red or pink. What makes the nodules pink? This is due to the presence of leguminous haemoglobin or leg-haemoglobin.

Nodule Formation

Nodule formation involves a sequence of multiple interactions between Rhizobium and roots of the host plant. Principal stages in the nodule formation are summarised as follows:

Rhizobia multiply and colonise the surroundings of roots and get attached to epidermal and root hair cells. The root-hairs curl and the bacteria invade the root-hair. An infection thread is produced carrying the bacteria into the cortex of the root, where they initiate the nodule formation in the cortex of the root. Then the bacteria are released from the thread into the cells which leads to the differentiation of specialised nitrogen fixing cells. The nodule thus formed, establishes a direct vascular connection with the host for exchange of nutrients. These events are depicted in Figure 12.4.

The nodule contains all the necessary biochemical components, such as the enzyme nitrogenase and leghaemoglobin. The enzyme nitrogenase is a Mo-Fe protein and catalyses the conversion of atmospheric nitrogen to ammonia, (Figure 12.5) the first stable product of nitrogen fixation. The reaction is as follows:

Figure 12.4 Development of root nodules in soyabean : (a) Rhizobium bacteria contact a susceptible root hair, divide near it, (b) Successful infection of the root hair causes it to curl, (c) Infected thread carries the bacteria to the inner cortex. The bacteria get modified into rod-shaped bacteroids and cause inner cortical and pericycle cells to divide. Division and growth of cortical and pericycle cells lead to nodule formation, (d) A mature nodule is complete with vascular tissues continuous with those of the root

The reaction is as follows:

The enzyme nitrogenase is highly sensitive to the molecular oxygen; it requires anaerobic conditions. The nodules have adaptations that ensure that the enzyme is protected from oxygen. To protect these enzymes, the nodule contains an oxygen scavenger called leg-haemoglobin. It is interesting to note that these microbes live as aerobes under free-living conditions (where nitrogenase is not operational), but during nitrogen-fixing events, they become anaerobic (thus protecting the nitrogenase enzyme). You must have noticed in the above reaction that the ammonia synthesis by nitrogenease requires a very high input of energy (8 ATP for each NH₃­ produced). The energy required, thus, is obtained from the respiration of the host cells.

Figure 12.5 Steps of conversion of atmospheric nitrogen to ammonia by nitrogenase enzyme complex found in nitrogen-fixing bacteria

(ammonium) ion. While most of the plants can assimilate nitrate as well as ammonium ions, the latter is quite toxic to plants and hence cannot accumulate in them. Let us now see how the is used to synthesise amino acids in plants. There are two main ways in which this can take place:

(i) Reductive amination : In these processes, ammonia reacts with α-ketoglutaric acid and forms glutamic acid as indicated in the equation given below :

(ii) Transamination : It involves the transfer of amino group from one amino acid to the keto group of a keto acid. Glutamic acid is the main amino acid from which the transfer of NH2, the amino group takes place and other amino acids are formed through transamination. The enzyme transaminase catalyses all such reactions. For example,

The two most important amides – asparagine and glutamine – found in plants are a structural part of proteins. They are formed from two amino acids, namely aspartic acid and glutamic acid, respectively, by addition of another amino group to each. The hydroxyl part of the acid is replaced by another NH2– radicle. Since amides contain more nitrogen than the amino acids, they are transported to other parts of the plant via xylem vessels. In addition, along with the transpiration stream the nodules of some plants (e.g., soyabean) export the fixed nitrogen as ureides. These compounds also have a particularly high nitrogen to carbon ratio.

Summary

Plants obtain their inorganic nutrients from air, water and soil. Plants absorb a wide variety of mineral elements. Not all the mineral elements that they absorb are required by plants. Out of the more than 105 elements discovered so far, less than 21 are essential and beneficial for normal plant growth and development. The elements required in large quantities are called macronutrients while those required in less quantities or in trace are termed as micronutrients. These elements are either essential constituents of proteins, carbohydrates, fats, nucleic acid etc.,and/or take part in various metabolic processes. Deficiency of each of these essential elements may lead to symptoms called deficiency symptoms. Chlorosis, necrosis, stunted growth, impaired cell division, etc., are some prominent deficiency symptoms. Plants absorb minerals through roots by either passive or active processes. They are carried to all parts of the organism through xylem along with water transport.

Nitrogen is very essential for the sustenance of life. Plants cannot use atmospheric nitrogen directly. But some of the plants in association with N2-fixing bacteria, especially roots of legumes, can fix this atmospheric nitrogen into biologically usable forms. Nitrogen fixation requires a strong reducing agent and energy in the form of ATP. N2 -fixation is accomplished with the help of nitrogen-fixing microbes, mainly Rhizobium. The enzyme nitrogenase which plays an important role in biological N2 fixation is very sensitive to oxygen. Most of the processes take place in anaerobic environment. The energy, ATP, required is provided by the respiration of the host cells. Ammonia produced following N2 fixation is incorporated into amino acids as the amino group.

Exercises

1. ‘All elements that are present in a plant need not be essential to its survival’. Comment.

2. Why is the purification of water and nutrient salts so important in studies involving mineral nutrition using hydroponics?

3. Explain with examples: macronutrients, micronutrients, beneficial nutrients, toxic elements, and essential elements.

4. Name at least five different deficiency symptoms in plants. Describe them and correlate them with the concerned mineral deficiency.

5. If a plant shows a symptom which could develop due to deficiency of more than one nutrient, how would you find out experimentally, the real deficient mineral element?

6. Why is that in certain plant deficiency symptoms appear first in younger parts of the plant while in others they do so in mature organs?

7. How are the minerals absorbed by the plants?

8. What are the conditions necessary for the fixation of atmospheric nitrogen by Rhizobium. What is their role in N2 -fixation?

10. Which of the following statements are true? If false, correct them:

(a) Boron deficiency leads to the stout axis.

(b) Every mineral element that is present in a cell is needed by the cell.

(c) Nitrogen as a nutrient element, is highly immobile in the plants.

(d) It is very easy to establish the essentiality of micronutrients because they are required only in trace quantities.