Nitrogen Fixing Wheat (Extended Version 1)

Created on 12 Jul 2017

Ugur Sevilmis

Research Institute

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Target is to use interfamilial asymmetric protoplast fusion under advanced sucrose and nitrate starvation between albino haploid wheat cell and haploid soybean microplast for obtaining nitrogen fixing wheat.

Cell fusion is a rapid in vitro cell engineering method in which two or more cells are assembled asexually to obtain hybrid cells with single or multiple nuclei.

As new genetic material (genome or exDNA) is added, these hybrid cells acquire new genetic and biological properties (Qu et al., 2011).

The first successful study in the field of somatic hybridization was made in 1972 (Carlson, 1972).

Subsequent successes have been achieved in rice, rape, tomato, citrus (Grosser & Gmitter, 1990), potatos (Orczyk et al., 2003) and in some other plant species.

Many of the disease resistance strains have been accessed through somatic hybrids (Collonnier et al., 2003).

Binsfeld et al. (2000) obtained Maximilian Sunflower (Helianthus maximiliani L.) or Giant Sunflower (Helianthus giganteus L.), which contain chromosomes from two to eight.

Helgeson et al., (1986) used somatic fusion to make potato (Solanum tuberosum) resistant to Potato Leaf Curl disease.

Protoplast fusion is frequently successfull between intra-familial species.

Protoplast fusion is rarely successfull between inter-familial species.

Szarka et al., (2002) conducted an intertribal hybridization study in which albino maize (Zea mays L.) and wheat (Triticum aestivum L.) mesophyll protoplasts were fused in the PEG medium and obtained hybrids with mixed cytoplasm.

Intergeneric asymmetric hybrids can be obtained by fusion between dicotyledonous Arabidopsis thaliana and monocotyledonous wheat (Deng et al., 2007).

New somatic hybrids could also be obtained with symmetrical and asymmetric fusion between the monocotyledonous species Festuca arundinacea and the dicotyledonous species Bupleurum scorzonerifolium (Wang et al., 2011).

Theoretically fusion is possible between completely different cells.

In symmetrical fusions, the entire genome of both parents fuse. But when two genomes are mutated, a phenomenon called "Gene Conflict" can occur because certain chromosomes reject one another.

In addition, this technique also places significant amounts of undesirable genetic material (Shankar et al., 2013).

In asymmetric fusion, a limited amount of a genome after genome breakdown is transferred to the fusion product (Xia, 2009). For example, symmetric somatic hybrids of Brassica napus and Lesquerella fendleri are sterile, while asymmetric hybrids are self-fertile (Skarzhinskaya et al., 1996).

To obtain asymmetric hybrids, the donor protoplast genome is fragmented before fusion. As a result, a limited amount of the donor genome is transferred to the fusion product. With this technique, some of the problems that often occur in symmetric fusions are avoided (Shankar et al., 2013).

Genomic fragmentation is provided by irradiation, use of microplast, or the use of metabolic inhibitors such as iodoacetamide.

Iodoacetamide inactivates cytoplasmic genomes while irradiation and microprotoplast production break down the nuclear genome (Yemets & Blume, 2008).

We are targeting to use two special kinds of evolutionary stresses under interfamilial protoplast fusion: Advanced sucrose starvation in between advanced nitrogen starvation stresses.

Why?

According to the endosymbiosis theory, mitochondria and chloroplasts emerged when free-living bacteria (prokaryotes) were introduced into eukaryotic cells by endosmibiosis.

It is thought that the mitochondria with its own DNA are origined from Rickettsiales  and chloroplasts are origined from nitrogen fixing photosynthetic cyanobacteria.

Findings that support endosymbiosis theory include: The formation of new mitochondria and plastids in a binary-like process like in bacterias. The inability of a cell to produce mitochondria or chloroplasts when they are taken outside the cell. Presence of transfer proteins called Porin in the outer membrane of mitochondria and chloroplasts like in the bacterial cell. Different from the cell nucleus, mitochondria and plastids contains single circular DNA similar to genomes of mitochondria and Rickettsia bacteria. Genomic comparisons indicate that cyanobacteria are the genetic root of plastids. Ribosomes of chloroplasts and mitochondria are similar to those found in bacteria. "Molecular machinery" in cell cytoplasm is unique to eukaryotes, but the molecular machines in mitochondria are similar to bacteria.

In endosymbiosis, many basic questions such as the nature of the host, the evolution of distinctive eukaryotic features, and the events leading to the transition from symbiotics to organelles are unanswered.

An example for the genetic diversity in a compartmentalized cell : A Paramecium, a ciliate, incorporates "Holospora obtusa" (a distant relative of rickettsial pathogens) as a bacterial food. In contrast to other bacterias, Holospora remains intact in vacoules and migrates to the host's nucleus. The bacteria divide and mature in the nucleus and eventually break apart and initiate a new cycle of infection in which the bacteria do not harm the host cell in any way.

In an extreme endosymbiosis of a dinoflagellate with a diatom, five different genomes (two nuclei, two mitochondria and one plastid) were detected in the cells of a single organism (Imanian et al. 2010).

Because of the private life cycle, bacterial endosymbiotics undergo a rapid evolutionary genomic evolution and are increasingly attributed to the host's functions.

These and similar examples show that eukaryotes are receptive or at least tolerant to extracellular gentel guests, which allocate many complex cellular constructs to endosymbiotic host processing (Lang & Burger, 2012).

A complete analysis of the eukaryotic genome reveals that eukaryotes with archaea-related information processing mechanisms and bacterial-related operational genes in metabolism are the most complex genetic mosaics (Lopez-Garcia and Moreira 1999).

Primitive endosymbiotic mitochondrial transformation also involves gene transfer from the guest to the host genome (Cavalier-Smith 2006).

Compartmentalization should play an important role in the self-organization of complex substances and in the evolution of living cells and organisms in this respect (Lehn, 2002).

If endosymbiosis theory is true, plants must be aphotosynthetic before chloroplasts settled in primitive plant cells.

What was the energy source used by the apotosynthetic plants? With a series of astronomical observations made in the early 2000s, it has been observed that some distant planets and astroids contain simple sugars (Hollis et al., 2000; Beltrán et al., 2008).

In the primordial world ecology, free sugars must also be present in order for DNA and RNA to appear (Jørgensen et al., 2012).

Primitive aphotosynthetic plants are also likely to use these free sugars as an energy source in the pre-endosymbiotic period.

The number of sucrose consumers in this primitive ecology may inversely be reduced by increased number of sucrose consumers, and aphotosynthetic plants may formed endosymbiosis with photosynthetic bacteria with a mutualist behaviour.

In this case, sucrose starvation must be the driving force of endosymbiosis.

Can endosymbiosis be reproduced artificially?

Can artificial endosymbiosis, which can be performed under advanced sucrose starvation, produce nitrogen fixing wheat?

In the case of sucrose starvation in modern plant cells, in the first stage, cell growth stops, energy is consumed. Then glycoproteins are digested, cell walls are thinned and holes in their surface grow, vacoules overgrow and organelles are pushed into cell walls and then cell defense system collapses. Cell death occurs if sucrose starvation persists (Morkunas et al., 2012).

Research on Arabidopsis cell suspensions revealed that cellular proteins rapidly disrupted in the case of sucrose starvation and cell growth ceased immediately. Cell divisions determined by cell number and biomass accumulation stops just after the onset of sucrose starvation (Morkunas et al., 2012).

In cells that have sucrose starvation, autophagy aims to obtain substrates for respiration (Chen et al., 1994).

All organelles may be destroyed during the advanced sucrose starvation. Plastids, ribosomes and endoplasmic reticulum are consumed relatively early; plasma membrane, mitochondria and peroxisomes are more resistant (Baker and Graham 2013).

The stage at which the cell defense system collapses may have critical importance to alter the functions of a cell.

Long-term starvation results in the synthesis of many specific proteins known as Starvation-Related Proteins (STP) (Tassi et al., 1992).

After 12, 24, and 48 hours sucrose starvation in rice cells, it is observed that the expression activity was inhibited by the 855 gene, which increased 867 gene expression activity (Wang et al., 2007).

Energy and other resources are needed to cope with abiotic and biotic stress conditions; For this reason, metabolic processes such as respiration slow down in the condition of sucrose starvation, the stressors of the plants are more susceptible to stress.

Horsfall and Dimond (1957) reported for the first time the link between low sugar content and increased susceptibility of plants to fungal diseases, known as Low-Sugar Diseases.

Furthermore, induction of defense in host plant cells consumes too much energy (Swarbrick et al., 2006).

Why using an albino?

Albinos does not have the ability to photosynthesize and is frequently emerged in in vitro cultures of microspores (Torp & Andersen, 2009).

If you use an albino wheat as a protoplast fusion partner, you will work with a cell without a chloroplasts. So, this will result with advanced sucrose starvation in wheat cell in a short period of time.

Albino wheat cells produced from wheat pollens are haploid (their genomes are fragmented and asymmetric).

These albino wheat cells will be starved for energy and will be undefenced.

 Being undefenced will change allorecognition.

Allorecognition is the ability of an organism to separate self from nonself. It is observed in all multi-cell phyla that it is necessary for life. On the simplest basis, even single-celled beings should be able to distinguish between food and non-food so that they can react correctly to invading pathogens and avoid cannibalism.

It is also vital for the species-specific sperm-egg interaction during fertilization to distinguish themselves from the foreign.

Allorecognition is also necessary for the evolution of cells to work together, as in multicellular organisms, which is a limiting factor in social parasitism (Czaran et al. ). Allorecognition also modifies the output of somatic fusion between different tissues (Holman et al. 2013).

Additionally mutualsim will function high under this condition to adopt these cells to life.

Albino cells will require energy and other fusion partner (haploid soybean female cells) will offer this capacity with its chloroplast related genes.
To increase the mutualistic symbiosis between fusion partners, advanced nitrate starvation will also be used in parallel.

Wheat cells will also be straved for nitrogen by keeping in N deficient condition opposite of soybean ; 

Can deficiency of sources will force different cellular genomes to tie up each other after protoplasms fused?

Mereschkowsky (1905) says that: "Think of a palm tree, living in peace with a water source, and a lion hiding in a nearby shrub, with all his muscles stretched, bloodthirsty, ready to jump on an antelope and slaughter ;Simbiyotik theory, with all its nakedness, lies in its deepest mystery, and reveals and illuminates the two fundamental principles of the palm tree and the lion's foreground: the palm is peaceful with its behavior, passive because it is a symbiosis, because there are many small workers, green slaves (chromatophores). The lion, on the other hand, must feed itself, imagine that the cells of the lion are filled with chromotophores, of course, rapidly lies in peace between the palm, feels full or needs some mineral water.

 

.....to be continued in version 2


 Agriculture
 Agrifood
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 Genetic Resources
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