Nitrogen Absorption in Plants (With Diagram)

Until recently, the reductive amination of 2-oxoglutarate catalyzed by the enzyme glutamate dehydrogenase has been considered as the main reaction of ammonia assimilation in plants. Now there is convincing evidence that most of the inorganic nitrogen available to a plant is incorporated into the amide amino group of glutamine via the enzyme glutamine synthetase (L-glutamate ammonia ligase— ADP, GS).

Subsequently this amide amino group can be transferred to the 2-position of 2-oxoglutarate yielding 2 moles of glutamate by glutamate synthase (Glutamine: 2-oxoglutarate amino transferase, GOGAT). The GS/GOGAT system has been established to be involved in the process of organic nitrogen interconversion taking place especially in developing germinating seeds during synthesis and mobilization of nitrogenous storage compounds.

This has been established by using N-labelled percursors and in recent studies, the introduction of inhibitors of the assimilation enzyme has proved very valuable to discriminate between the GS/GOGAT and the GDH-pathway.

In heterotrophic tissues, apparently the intracellular distribution of the enzyme involved in nitrogen assimilation corresponds to the pattern observed in green tissues.

In root apices of young peas, nitrate reductase was found in the cytosol, nitrite reductase and GOGAT are restricted to the plastids and GS shares a location between cytosol and plastid; GDH was only detected in the mitochondria though in other heterotrophic tissues, it is also reported from plastids.

Most enzymes from heterotrophic tissues are active with both nicotinamide nucleotides, and seem to prefer NAD⁺ as also reported in pollen grains. Figure 11-5 shows summary of nitrogen fixation.

Sulphate Metabolism:

Sulphur is one of the six major elements of which most biochemical substances are composed of. These substances include glutathione, thiamine-pyrophosphate, biotin, lipoic acid, coenzyme A, non-heme iron proteins, and those proteins which contain cysteine and methionine. The ultimate source of sulphate for most of the organism is inorganic sulphate.

There are also structures like mercaptane which are found in the roots of radish or sulphides or disulphides, polysulphides or sulphoids or in garlic oil as methyl sulphonium compound. Some of the most important sulphonium compounds are methyl methionic and S- adenosyl methionine. Their main function is to transfer methyl group to other compounds.

Sulphate enters two kinds of reactions mostly esterification and reduction. It may be mentioned that the former reaction is very common in prokaryotes and eukaryotic microorganisms but is scanty in higher plants. In red algae sulphated polysaccharides are reported in cell walls. In the reduction reactions, sulphur in reduced to the thiol level.

This form of biochemical reaction is most prevalent in higher plants and other organisms except animals. In nature two kinds of processes are reported and these are assimilatory (formation of cysteine and methionine) and dissimilatory (H S formation). In the former sulphur is reduced to thiol form and this is trapped by the organic acids giving rise to some sulphur containing amino acids.

In the latter reaction sulphate reduction is accompanied by the release of energy which is utilized in ATP formation from ADP and inorganic phosphate. Most studies suggest that sulphate uptake is an active process and is transported through a carrier which is possibly an enzyme.

The uptake of sulphate is inhibited by several compounds including methionine, cysteine, sulphate and thiosulphate etc. Sulphate reduction is an endergonic reaction where 14 Kcal of energy is utilized. The energy is obtained from ATP possibly through an enzyme ATP sulphurylase.

Sulphate Metabolism:

Sulphate reacts with ATP and forms adenosine-5′- phosphate sulphate (APS) as shown below:

The first reaction overcomes about 9 kcal of the 14 kcal energy barrier of the reduction of sulphate and thus can move in the reverse direction. APS kinase enzyme makes 5 kcal when APS reacts with ATP to form phosphoadenosine phosphate sulphate (PASP).

Evidently in the three enzymatic reactions one molecule of sulphate is used up and two molecules of ATP are utilized and thus endergonic sulphate reaction continues to proceed in the forward direction. The three enzymes are ATP sulphurylase, APS kinase and pyrophosphatase. Then sulphate which was activated is reduced to sulphide in four steps and each of the steps utilizes two electrons or hydrogen atoms:

H₂ SO₄ + 8 (H) → 4H₂O + H₂S

Sulphatereductase which is made up of A and B enzymes and a protein fraction acts upon active sulphate (PAPS) and NADPH₂ supplies the requisite electrons. The precise function of the two enzymes is conjunctural. Enzyme A possibly reduces a disulphide bond in fraction C and enzyme B mediates reduction of PAPS to sulphite. Six more electrons are required to reduce sulphite to sulphide (Fig. 11-7).

Several of the organisms have been shown to have specific sulphotransferases which catalyze the esterification of different organic compounds with sulphate or sulphite. Accordingly specific types of sulphate esters and sulphonic acids are formed. It is generally assumed that sulphated polysaccharides in the cell walls bring about enough protection against desiccation.

In the sulphonic acids sulphur is present in a more reduced state than in sulphated compounds. Some of these are cysteic acid, glucose- 6-sulphonate. It may be added that in sulphonic acid sulphur is directly linked to carbon whereas in sulphate esters an oxygen atom is present between carbon and sulphur.

In plants and bacteria, the major pathway proposed for the S^2- to cysteine conversion utilizes the carbon skeleton of serine which is first activated by conversion to an O-acetyl derivative. Cysteine is then the source of sulphur in the biosynthesis of methionine. The transfer of sulphur involves the condensation of cysteine with O-succinylhomeserine to yield cystathionine.

In recent years much research is being directed towards the understanding of mechanisms of prokaryotic nitrogen fixation at metabolic and molecular levels. Most studies have been conducted with legume-Rhizobium system where several methods for the increase of nitrogen fixation have been proposed.

Some of the possibilities are: legumes development having high photosythetic rate since amount of photosynthate available to the legume limits the amount of nitrogen fixed by the bacterium. Number of nodules could be increased and this would enhance nitrogen fixation by a plant.

Hardy and his associates have demonstrated that high carbon dioxide concentrations over soyabean fields enhanced their yield due to high rate of photosynthesis and thus high nitrogen fixation.

Another possibility is to select Legume- Rhizobium associations which do not waste energy by evolving hydrogen. Two plant species have also been reported and these are cowpea and alder. Yield could be enhanced if such a mechanism could be transferred from these two species in other legumes.

Third and most investigated possibility is development through genetic manipulation of Rhizobium strains which are insensitive to soil ammonia and nitrate concentrations. Such strains would be able to fix nitrogen continuously even though immersed in a high level of nitrogen.

This could be used effectively as a source of nitrogen fertilizer. Selection of bacterial strains which could inhabit cereal roots and provide sufficient fixed nitrogen for normal plant growth is needed. Most studies are centered around transfer of nitrogen- fixing genes to a harmless bacterium which would invade plant cells and transfer nitrogen fixing system similar to nodules.

Some attempts on direct transfer of nitrogen fixing genes to the higher plants are also being examined. Thus, the plant would be able to fix nitrogen directly from the atmosphere. We shall briefly mention some progress made in the field of genetic engineering with nitrogen fixing bacteria and leguminous plant cells.

Recent studies have shown that Rhizobium can fix nitrogen itself under culture conditions and the presence of legume was not specifically required. Some workers have even succeeded in establishing soyabean cells and Rhizobium symbiosis under in vitro conditions. In 1975 Child and LaRue reported that rhizobia specific for cowpea could fix nitrogen when cultured with wheat or tobacco.

It is generally suggested that some signals from the cultured plant cells trigger nitrogen fixation in rhizobia. However, recent studies discount such a suggestion. It has been shown that pentose sugar and dicarboxylic acid are required for nitrogen fixation by the Rhizobium.

Evidently the microorganisms had the genetic information needed for the biosynthesis of nitrogenase. Some attemps on the transfer of ‘nif’ genes from the nitrogen fixing bacteria to non-nitrogen fixing bacteria have also been made. In fact nif genes from Klebsiellapneumoniae to Escherichia coli have actually been transferred. A few hybrid cells resembled Klebsiella in their nitrogenase activity.

Further evidence was provided regarding the transfer of control genes and nif genes to E. coli. Subsequently nif genes from E. coli were transferred to a plasmid which carried a sex factor. This was called F’-nif.

Such an association enabled transference of nif genes from E. coli to other bacteria through conjugation. Very recently nif genes of K. pneumoniae have been transferred from F’-nif factor to another plamid RP₄ with a view to construct even more transferable plasmids.

Using the latter plasmid it was possible to transfer nif genes between two very different classes of bacteria. Nif genes from K. pneumoniae were transferred to mutants of Azotobactervinelandii which has no capacity to fix nitrogen.

It may be added that such advances are highly significant since Klebsiella is an anaerobic organism and nif genes normally function only in the the absence of oxygen. Azotobactervinelandii can fix nitrogen under aerobic conditions. Similarly other workers have reported the transfer of nif genes from Rhizobium trifolii to Klebsiellaaerogenes 418. The latter strain is not capable of fixing nitrogen.

Here the transfer was mediated by F-like R factor. Comparatively very few studies are reported on successful transfer of nif genes into higher plant cells. Gene transfer to higher plants is a normal feature for Agrobacterium tumefaciens which produces crown gall disease in higher plants by permanent incorporation of its plasmid genome in the host genome. Available studies have shown that nif gene of Rhizobium is plasmid-borne.

When rhizobia are treated with acridine dyes they lose their ability to form nodules. Recently Nuti (1979) in a short communication in Nature have reported hybridization between restriction fragments of plasmid DNA from Rhizobium leguminosarum and DNA encoding part of the structural gene for Klebsiellapenumoniaenitrogenase.

Clearly at least some of the structural genes for Rhizobium leguminosarumnitrogenase are plasmid-borne.

It needs also mentioning that overlapping sequences of K. pneumoniae DNA have been cloned on small amplifiable plasmids.

These techniques could be further exploited to prepare physical maps of the Rhizobium nitrogenase genes analogous of K. pneumoniae. In fact cloning of nif genes will help in the purification of large amounts of nif genes for biochemical genetic investigations and will furnish material for the construction of genetic material which would replicate and express nif genes in the plant and bacterial cells.

Protoplast fusion methodology is also being utilized to explore the possibility of nif gene transfer. Blue green algae and nitrogen fixing bacteria protoplast fusion is already being attempted. Perhaps another approach is to fuse the protoplast of leguminous and non-leguminous plants with a view to making non-legume species as a suitable host for the functioning of nitrogen fixing symbiotic bacteria.

However, much remains to be accomplished in this technology. The whole problem is highly complex and transfer of nif genes is beginning of the whole chain of events. Once introduced the alien genes have to seek acceptability and then produce functional products.

The major hurdle is the kinetics attribute of nitrogenase which is highly sensitive to oxygenase. The supply of energy is yet another problem to be solved. After all the plant cannot afford to divert all or most of its energy for nitrogen fixation. Once the rate of photosynthesis is increased this problem could be partially overcome.

At any rate when these experiments become the reality it should be possible to engineer use of fertilizers. In view of rocketing prices and oil crisis, the ushering in of era of’self fertilising farming’ may save the humanity from general economic crisis.

Cellular Energy Cost of N₂ Fixation:

Energy is used for N₂ reduction and also assimilation of NH₄⁺ within the plant cell cytoplasm and also for uptake of carbon compounds from the plant cell cytoplasm. Under optimal conditions, the purified nitrogenase-nitrogenasereductase complex requires a minimum of two molecules of ATP for each electron transferred to a reducible substrate.

ATP hydrolysis is associated with transfer of electrons from nitrogenasereductase to nitrogenase. Nitrogenase for each N₂ reduced requires 16 molecules of ATP. Further H₂ evolution by nitrogenase requires a significant amount of energy. In most legumes 40 to 60% of the eletrons used by nitrogenase were allocated to protons for H₂ production with N₂ as substrate.

It is estimated that reoxidation of one mole of H₂ by equivalent could generate 2 moles of ATP by oxidative phosphorylation of equivalent reductant. In a recent review, Phillips has given whole-plant energy cost of symbiotic N₂ fixation in nodulated legumes.

The data are expressed in terms of carbon utilization. Thus for Pisumsativum g C/gN value was 6.8 (measured through reduction ratio C₂ H₂ / N₂ = 3.02 and for Glycine max it was 6.3. In such estimates CO₂ evolution and C₂H₂ reduction of intact nodulated roots with varying C₂H₂ reduction method was employed.

Some reviews have also appeared where possibilities of enhancing N₂ fixation in legumes have been widely discussed and these are summarized below:

(i) Establishing functional root nodules earlier.

(ii) By selecting host-plant phenotypes as well as Rhizobium mutants.

(iii) Legume germplasm collections may afford other genetic traits that alter symbiotic N₂ fixation.

Transport and Partitioning of Nitrogeneous Solutes:

Pate (1980) has reviewed recent information on the identity of the nitrogenous compounds which and transported in the long-distance through channels of higher plants.

He has especially discussed the flow profiles for carbon and nitrogen in xylem and phloem of a plant.

By analysing the conducting elements of xylem and phloem it has been shown that main pathways for long-distance transport of nitrogeneous solutes in vascular plants are mass flow through xylem from root to regions of transpiration in the shoot, and flow of translocate in phloem from photosynthetically active organs to sites in which photosynthate is used for growth or storage.

Levels of N in xylem sap lie usually in the range 0.01 to 0.21% (w/v) while in phloem sap the concentration is 10-20 times this range. Xylem fluid has a C:N weight ratio usually within the range 1.5 to 6 indicating importance of nitrogen-rich compounds in xylem transport. In phloem C:N ratio ranges from 15 to 200.

The reaction of xylem sap is acidic whereas that of phloem is alkaline. Nitrate, but not nitrite, is a common constituent of xylem exudates of many species, especially when high levels of nitrate are available to the roots.

In phloem exudates nitrate is usually absent or present in very low levels. In general a positive correlation is noticed between the in vivo or in vitro level of nitrate reductase of the root and the ratio of organic-N: NO₃ sup in xylem exudates.

Roots of Cucumis, Gossypium have weak NR activity in roots and over 95% of the xylem N may consist of free nitrate. Roots of such plants synthesize and export amide-N if given urea or ammonium suggesting high NH₄⁺ assimilation but limited NO₃^‑ reduction. Very little information on the patterns of N uptake and assimilation by woody species exists.

Cloning of genes for several protein transporters:

Pertinent selection strategies have been devised to isolate transport mutants. Nitrate transport mutants or mutants for nitrate reduction have been selected by growth in the presence of chlorate (CIO₃^–). Chlorate is a nitrate analog that is taken up and reduced in wild type plants to the chlorite which is a toxic product.

Plants resistant to chlorate, if selected are likely to show mutations that block nitrate transport and reduction. In Arabidopsis several such mutations have been isolated. The first transport gene encodes a low- affinity inducible nitrate-proton symporter.

At least four separate genes are involved in nitrate uptake and two are constitutively expressed genes for low and high affinity uptake respectively, and two nitrate- inducible genes for low and high- affinity uptake, respectively.

Further studies have cloned some plants transport genes by identifying regions of sequence similarity with transport ganes of other organisms, and occasionally they have been able to identify the gene after purifying the transport protein.

Many yeast transport mutants are known and are used to identify corresponding plant genes through complementation. In this way it has been possible to identify several inward-rectifying K⁺ channels. Further, of the inward-rectifying K⁺ channel genes identified so far, one is expressed strongly in stomatal guard cells, another in roots, and a third in leaves.

Another gene from wheat has been identified for high-affinity active transport of K⁺. This gene was expected to encode a K⁺-proton symporter, but it carries Na⁺ instead of K⁺, with the K⁺ ions.

Similarly genes for plant vacuole H⁺ – Ca²⁺ antiporters have been isolated. Another protein which seems to form water channels in membranes have been named aquaporins and appear to be regulated by protein phosphorylation, in response to water availability. The general assumption is that family of genes rather than an individual gene exists in the plant genomes for each transport functions.