Nitrogenomics: Is the term worth science?

K.K.Vinod

Anyone searching the internet for the term 'nitrogenomics', chances are that you will be taken to a page describing,

Nitrogenomics as the branch of the study of genomics pertaining to nitrogen utilisation and assimilation in organisms. Nitrogen is a primary nutrient essential for sustaining the life of every organism. Nitrogen is freely available in the atmosphere and Earth's crust and is generally assimilated by plants and microorganisms, then moved to higher organisms through the food chain. Genomics of nitrogen assimilation lie at different levels of organisms, from microbes to higher organisms, where different genetic controls regulate the actual assimilation.

This term nitrogenomics was not available before April 2004, when I made the posting of this term for the first time in Wikipedia. The growing awareness of genomic sciences and their enormous application potential, led me to think of a specialised term for the molecular genetics and genomics of the nitrogen utilisation in the organic world. It rather amused me that the words 'nitrogen' and 'genomics' fused well. Though I coined the term without much thinking of its potential as a branch of science, I now feel that the science which is described within this term can be the science of life itself.

Nitrogen is as essential as carbon, hydrogen and oxygen in the organic world, as it forms the primary component of amino acids which makes proteins, the building and guiding blocks of organic life. But the most intriguing part of the story is that nitrogen is not freely available as in the case of other primary elements of life. In fact it is mostly available abundantly in the unavailable forms. So how then nitrogen comes into life? Its an intricate cycle that involves enormous microorganisms, entire plant kingdom and the whole animal kingdom or every living organisms in interaction with the forces of nature! The science behind this is immense.

I learned the indispensable requisiteness of this science when I started working on the genome mapping of nitrogen assimilation genes in our staple food crop, rice. By this time, more than one and a half centuries had passed since we started adding nitrogen fertilisers into agriculture. More and more nitrogen added gave more and more food grains, enabling us to feed millions of mouths that were born in the world. Indiscriminate fertiliser usage had started to take its toll through irreversible damage to the ecosystems, nitrate poisoning, eutrophication of the water bodies so on and so forth... How are we going to cope up to this situation? One way is by regulating the usage of inorganic nitrogen sources while we look for better living environment with sufficiency of food. Here we require the science I am talking of....nitrogenomics. I am convinced...are you?

My Professor's Dilemma

K.K.Vinod and M. Maheswaran

Yesterday my Professor, Dr M Maheswaran wrote me after reading the book “Emperor of All Maladies- A Biography of Cancer” by Siddhartha Mukherjee.

He wrote,

Genes talk to genes and pathways to pathways in perfect pitch, producing a familiar yet foreign music that rolls faster and faster into a lethal rhythm.

If the data did not fit the dogma then the dogma, not the data, needed to be changed.

After reading the book and understanding the biological causes behind the cancer genetics I felt very bad about things what we are teaching and doing in understanding the genetics of many of the traits we deal with crops and exploit the results in practical plant breeding. Many of the concepts of predictions we make based on the classical genetics, whether it is Mendelian or Galtonian, remain irrelevant considering the biological implications on a particular phenotype. For example, p53 gene, an unassuming name, has major role in the development of human cancer than any other component of the genome. The gene get its name from the product it encodes, p53, which is a polypeptide having a molecular weight of 53 kilo Daltons. p53 was thought for number of years to be a dominantly acting oncogene, but in 1990, it was recognized as the tumour suppressor gene that, when absent, is responsible for a rare inherited disorder called Li-Farumeni syndrome, whose victims are affected with a very incidence of certain cancers, including breast cancer and leukaemia (though there are separate and specific genes for breast cancer and leukaemia). Like individuals with the inherited form of retinoblastoma, persons with Li-Farumeni syndrome inherit only one functional copy of the p53 tumour suppressor gene and are thus highly susceptible to cancer as the result of random mutations that knock out the function of the remaining copy of the gene. Further, if we see the development of cancer in humans (for any type of cancer development), the possible sequence of genetic changes in a cell lineage are given below.

 

The complexity of cancer can be better understood if you read the above book. But in plant genetics we decide the genes based on the prediction methods and most of the gene predictions are based on the “breeder friendly phenotyping methods (whatever be the trait we have the simple means and many models to predict the genes). The advent of molecular marker technology made this simpler. Once there is co-segregation of DNA marker, the gene discoverer assumes he/she got gene for the phenotype (some where I read that there are 83genes identified for resistance to rice blast disease and we have 26 genes for brown plant hopper resistance in rice- just go back and read the situation of gene for Li-Farumeni syndrome). Gene identification based on breeder friendly phenotyping and pyramiding of those genes without knowing the functionality of the genes is a wasteful exercise. In recent times people started asking questions whether it is good to pyramid dominant genes or recessive genes without understanding the situation of obscurity prevailing over dominance or recessiveness. This can be remedied if every gene discoverer realizes the importance understanding the biology behind each of the phenotypes instead of evolving prediction methods.

My Professor stops his letter by saying “Predictions are always based on perceptions”.

Concern of my professor was of a genuine teacher, who attach paramount importance to imparting current and competitive knowledge to his students. He laments that except a very few teachers of plant genetics, none are ready to change the way science unfolds life’s mysteries. So instead of changing the dogma, we are often bending the data fit the dogma. Are we ruthlessly incompetent because we are afraid of deviating from the conventions?

UV-B Radiation Stress in Plants

K.K.Vinod

Our nearest star, the sun, emits short wavelength radiation that is incident on the earth’s atmosphere. Most of the radiation in the atmosphere is infrared radiation (700-3000 nm, 67% of the photons) and visible light (400-700 nm, 28%; Nobel, 1983). Ultraviolet radiation (UV, 200-400 nm), on the other hand, reaches the atmosphere in smaller amounts (5% of the photons). The biologically most hazardous part of UV radiation, i.e. UV-C (200-280 nm) and UV-B (280-320 nm) below 290 nm, are completely absorbed by the stratospheric ozone (O₃) layer and by other oxygen molecules in the atmosphere (Frederick, 1993). In addition, the ozone layer absorbs some longer-wave UV-B and UV-A radiation (320-400 nm) (Fig. 1). Consequently, of the photons at the earth’s surface, only about 2% are in the ultraviolet range (Nobel, 1983). However, of the total solar energy reaching the earth’s surface, UV-B radiation comprises about 1.5% and UV-A radiation about 6.4% (Frederick et al., 1989). The intensity of UV-B radiation, in particular, is affected by the thickness of the ozone layer, which in turn varies periodically as a consequence of natural processes such as seasons, winds and solar cycles. In addition, latitude, time of year and time of day determine the length of the path of a UV-B photon through the absorptive ozone layer (Caldwell et al., 1980). 

Fig 1. Ultraviolet spectrum 

On average, the ozone concentration in the stratosphere is low, i.e. about ten ozone molecules per million molecules of air; and it is highly dynamic because the ozone molecules are created and destroyed continuously. However, since the 1970’s, human activities have disrupted the natural balance between synthesis and breakdown of ozone. Depletion of the ozone layer has repeatedly been reported to occur over Antarctica, but in the 1990’s there were also frequent occurrences of major spring-time ozone depletion over the Arctic. It has been found that the main man-made compounds responsible for enhancing ozone breakdown are the chlorofluorocarbons (CFC) (Fig. 2) and nitrogen oxides. Recently, it was also found that the increasing concentrations of greenhouse gases result in stratospheric cooling, thus creating suitable conditions for breakdown of ozone molecules. Therefore, the most recent predictions based on stratospheric chemistry and climate-change models estimate that in the northern areas (60-90° N), compared with the long-term means, the maximum springtime UV-B radiation will increase up to 50-60% in 2010-2020. 

Fig 2. Chlorine atoms induce the decomposition of two ozone molecules into three oxygen molecules in a net chain reaction in which the chlorine atoms are regenerated so that decomposition of ozone continues. 

Effect of UV-B radiation in plants

Elevated levels of UV-B radiation will have many direct and indirect effects on plants (Fig. 3). Even present-day levels of UV-B radiation affect the growth and development of plants. The direct effects of UV-B radiation on plant cells are mostly damaging, because UV-B photons have enough energy to create lesions in important UV-B-absorbing biomolecules such as nucleic acids and proteins. It is known that the photoproducts of DNA formed by UV-B radiation, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone and (6-4) photoproducts, are all toxic and mutagenic. In addition, the altered DNA and RNA structures may interfere with transcription and replication; and therefore protein synthesis may be slowed down during UV-B stress. In order to avoid the effects of DNA damage, plants have efficient systems for DNA repair, including photoreactivation and excision repair, which are involved in restoring the structure of genetic material during exposure to UV-B radiation. However, the indirect effects of UV-B on plant cells can also be damaging: UV-B radiation may cause oxidative damage in chlorophylls and polyunsaturated lipids by increasing the formation of free radicals and peroxides (Jordan, 1996). To prevent oxidative damage, cells contain antioxidants, e.g., phenolic compounds, that scavenge the free radicals. Phenolic compounds have variable antioxidant properties; and several studies have shown that during UV-B exposure, the production of compounds with efficient antioxidant structures, such as additional hydroxyl groups on ring B of the flavonoid skeleton, is favoured. Plant cells also contain enzymes, e.g., superoxide dismutase (SOD) and catalase, which scavenge superoxide radicals and protect the cells against H₂O₂, respectively. 

Fig. 3. Effects of UV-B radiation on plant system.

Signal transduction and gene expression 

In addition to damaging plant cell components, UV-B radiation often exerts its effects through altered patterns of gene activity; e.g., the effects of UV-B radiation on photosynthesis, UV-B-screening phenolics, growth, reproductive processes, plant form and timing of life phases, are all caused by altered gene action. The mechanisms by which plants perceive UV-B radiation are not fully understood, but it has been suggested that direct absorption of UV-B by DNA could result in the formation of a “signal” that regulates the transcription of genes.

Signal transduction pathway in relation to UV-B radiation stress. Reactive oxygen species (ROS) increase in response to UV-B and are an important component in the regulation of both up-regulated and down-regulated genes. The nature and origin of the ROS involved in the early part of UV-B induced signalling pathways have been investigated in Arabidopsis thaliana. The increase in PR-1 transcript and decrease in light harvesting chlorophyll binding gene (Lhcb) transcript in response to UV-B exposure was shown to be mediated through pathways involving hydrogen peroxide (H₂O₂) derived from superoxide (O₂^•-). In contrast, the up-regulation of PDF1.2 transcript was mediated through a pathway involving O₂^•-- directly. The origins of the ROS were also shown to be distinct and to involve NADPH oxidase and peroxidase(s). The upregulation of CHS by UV-B was not affected by ROS scavengers, but was reduced by inhibitors of nitric oxide synthase (NOS) or NO scavengers. Together these results suggest that UV-B exposure leads to the generation of ROS, from multiple sources, and NO, through increased NOS activity, giving rise to parallel signalling pathways mediating responses of specific genes to UV-B radiation (Fig. 4).

In addition, it has been hypothesized that in plant cells, specific UV-B photoreceptor-mediated signalling processes regulate gene expression. However, the characteristics of a UV-B photoreceptor and how the signals are transduced after UV-B perception, are not yet known.

In addition to the increase in ROS other known signal transduction intermediates increase their levels. These include salicyclic acid (SA), jasmonic acid (JA) and ethylene. Using Arabidopsis mutants that are insensitive to SA, JA and ethylene (NahG, jar 1 and etr 1-1 respectively), clear differences in gene activity response have been demonstrated. For instance, an increase in expression of the two pathogen-related genes PR-1 and PDF1.2 are depended on SA and ethylene or JA and ethylene respectively. In contrast, down-regulation of RNA transcripts for photosynthetic proteins was independent of all three compounds. Furthermore, although ROS is involved in down-regulation of RNA for photosynthetic proteins, the chloroplast signal may not be involved.There are at least three separate signal transduction pathways involved in UV-B gene regulation and substantial “cross-talk” must take place.

A unique response of plants to UV-B radiation relates to the property of high photosynthetically active radiation (PAR) to ameliorate its damaging impact. The interaction of UV-B and PAR was initially discovered at the physiological level, but Jordan et al., (1992) demonstrated that high PAR also reduced down-regulation of gene expression. This ‘protection’ against UV-B damage did not involve synthesis of protective pigments, but was related to the function of the photosynthetic apparatus itself. The photosynthetic system can act as a photoreceptor and specific wavelengths can change chloroplast gene expression. Research conducted by Jordan et al., (1994) on etiolated tissue is also indicative of a strong link between the development of the photosynthetic apparatus and UV-B-induced gene expression. The connections between UV-B radiation and photosynthesis, and the signal transduction pathways that lead to modification of gene expression are yet to be comprehended.

Fig.4.  Schematic illustration of signal transduction pathways induced by UV-B radiation

It was found that in genetically modified tobacco (Nicotiana sylvestris) increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin indicating that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.  

References

Caldwell, C.R. 1993. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L.) microsomal and soluble protein tryptophanyl residues in vitro. Plant Physiology 101:947-953.

Frederick, J.E. 1993. Ultraviolet sunlight reaching the Earth’s surface: a review of recent research. Photochemistry and Photobiology 57:175-178.

Nobel, P.S. 1983. Biophysical Plant Physiology and Ecology. Freeman & Co., New York, pp.185-238.

Jordan, B.R. 1996. The effects of ultraviolet-B radiation on plants: a molecular perspective. In: Callow, J.A. (editor). Advances in Botanical Research, Vol 22. Academic Press, New York, pp. 97-162.

Jordan, B.R., P. James and S.A-H. Mackerness. (1998). Factors affecting UV-B induced changes in Arabidopsis thaliana gene expression: role of development, protective pigments and the chloroplast signal. Plant & Cell Physiolog, 39: 769-778.

Jordan, B.R., J. He, W.S. Chow and J.M. Anderson. Changes in mRNA levels and polypeptide subunits of ribulose bisphosphate carboxylase in response to supplemental UVB radiation. Plant, Cell and Environment 15: 91-98, 1992.

Why I am proud to be a Plant Breeder?

Plant breeding was the science which existed as long as the humans did... It probably started when the first man was hungry.. and when he selected what to eat... and when he probably thought to have his favourite food near his home.... He did exploration, collection and conservation of the grass and grains he needed... he grew the fruits he was fond of... He did selection of the best among the natural variability...Later, he started agriculture...and finally, selecting those types which yielded more than the one he was growing earlier.

PLANT BREEDING was continuous process...Earlier man never knew how variation occurred...and never knew how it was occurring... and how it perpetuated.

The science of GENETICS opened up a whole new area of knowledge.. the knowledge which catered the needs of ever growing global population.. The knowledge which is going to sustain the generations to come..

COME...LET US PARTAKE THAT GREAT KNOWLEDGE