Novelty: PLASTID MUTATIONS
N.Shemorakov, Kaluga region
Plastids consist of specific proteins, DNA and RNA. Depending on the type of pigments plastids are divided into chloroplastids (green pigment), chrysoplastids (yellow or brown), pheoplasts (fulvous), rhodoplasts (red) and leucoplasts (colorless).
The quantity of plastids per cell may vary from 1 to 400 and more at different kinds of the supreme and lowest plants.
The number of plastids per cell depends on its specialization, age, location and other factors.
The inner structure of plastids is complicated. There is a membrane system, a type of endoplasmatic reticu-lum.
The DNA of plastids differs from the DNA of cell chromosomes in the contents of nitrogen base. And the RNA of plastid ribosome differs from the ribosome RNA of cytoplasm. The genetic information of plastids is in plastid DNA.
It is established that plastids are reproduced by cut-cross division and go to the daughter cells during mitosis and meiosis.
The cells that lost plastids can not make them again. Thus, single-celled
Euglena mesmily has about 100 plastids, but if it is kept in the darkness the reproduction of plastids is suspended and while the cells divide there can appear those without plastids at all and unable to reproduce them. In the cul-ture they can exist only on nutrient substance.
Some authors consider the presence of plastids in vegetable cells as symbiosis between the animalcular uni-cellular alga and supreme plants.
Plastids in the plant cells are absolutely autonomous.
The cytoplasm of the cells of plants is the biotope for plastids. They adapt or die there. There is natural se-lection of plastids in the cytoplasm. Supreme plants unwillingly depend on the plastids' activity because if a seed of a plant germinates in a poorly lighted place chloroplasts will not be able to produce photosynthesis and the seedling will not be able to produce intracellular biosynthesis and thus it is doomed to die. The plants seek the light "to obey chloroplasts". The side of the plant not lit by direct light grows more quickly than the one lit. It happens in spite of the fact that biosynthesis goes on more active in the cells of the lit side.
In that case the plant "obeys chloroplasts" again, it tries to "light" the other part of cells. Plants developed this ability during the evolution. The individuals not having this quality died without leaving posterity. The indi-viduals having this quality left posterity with the same quality, and the ones with abnormality died in the process of the natural selection. The plant extends to the light despite its intensity. This process has a very complicated in-teraction mechanism of plants and plastids cells.
The vital functions of plastids depend not only on sufficient quantity of sunlight but also on the chemistry and the physical condition of the cytoplasm of the cell.
Similar to the cells of supreme plants plastids can function and reproduce only in the optimal conditions. In the case of certain genotype of cellular nucleus cytoplasm may have the chemistry unacceptable for normal func-tioning of all the types of plastids. In this case plastids can not synthesize one or several types of pigments, e.g. chlorophyll A and xanthophylls, and we can witness the phenomenon of low chlorophyll and even absence of chlorophyll.
In the optimal light regime there is certain balance of chloroplasts, chromoplasts and leucoplasts characteris-tic of taxon. If the light regime changes the balance changes too. Plastids react keenly upon the quantity of light, its intensity and spectral distribution. If the portion of ultraviolet rays goes up the plant turns red.
What goes on? A part of plastids in the epidermis cells begins to synthesize carotinoids. If the portion of ultra-violet is not significant they can synthesize both carotinoids and chlorophyll. The quantity of carotinoids depends of the intensity of ultraviolet. But plastids containing a large quantity of carotinoids (chloroplasts) do "a favor" to the plastids under them. Chloroplasts consume ultraviolet rays, filter, disperse and refract them and thus decrease the intensity of their influence over the below lying plastids.
As soon as the intensity of light decreases the quantitative ration of plastids of different colors returns to the initial relative balance. Thanks to such an ability of plastids the plant can "stretch" to the source of light in spite of its intensity. Of course, this ability is not unlimited. After overpassing a certain threshold of light intensity the in-tensity of photosynthesis decreases. That results in the stop of plant's growing and even its death.
The question is: how do plastids react to physical and chemical irritants? How does it happen that the in-creasing concentration of salts in cellular sap or growth of light intensity makes plastids produce other pigments instead of chlorophyll?
The most probable answer is that nucleic acids of plastids (DNA and RNA) are very sensitive both to physi-cal and chemical irritants, and these are they who change the direction of synthesis inside plastids. That means that to change biosynthesis inside a plastid its DNA and RNA should change themselves. Moreover these changes should be reversible because the environment, both physical and chemical, changes periodically.
If DNA changes are irreversible it will result in particular specialization of such a plastid, that means that it will be able to exist and function only at certain conditions of both internal and external environment.
The condition of intracellular environment changes time to time, and one of the changes can cause the death of plastid. But in most cases it does not happen. That means that at physical and chemical impacts DNA changes are reversible. These changes can not be called typical mutations.
The changes of transport and information RNA of plastids can be both reversible and irreversible. It does not matter a lot for plastids as these RNA can be synthesized again being changed according to plastid DNA.
Ultra-violet rays can excite electron shells which results in forming photoelectrons that give rise to different chemical reactions leading to mutations. (M.E. Lobashov, 1967). Thanks to the length of the wave ultra-violet rays penetrate badly into the inner tissues of plants. Ultra-violet rays are most effective in mutations of unicellular or-ganisms as they easily reach the nucleus of the cell.
So, what are plastids? They are unicellular nuclear-free organisms (prokaryotes) inside vegetable cells. Plastids in epidermis and subepidermis are subject to mutagen action of ultra-violet rays.
At present the mutagen action of ultra-violet rays is proved for all the organisms whose DNA can be directly exposed to rays.
Plastids are not exception as cellular and plastids shells are a weak obstacle for ultra-violet rays.
The most mutagen action of ultra-violet rays is connected with waves of 2500 -2800A. The fact is that nu-cleic acids absorb waves of this part of spectrum that is about 2600 A. The rays of this length destroy DNA most effectively and apparently this explains the high sensitivity of nucleic acids to them. Perhaps this is why the waves of this part of spectrum have the most bactericidal effect (M.E. Lobashov, 1967).
Ultra-violet rays are referred to electromagnetic oscillations but they do not give rise to ionization, their in-fluence on organisms consists in appearing of excited molecules and atoms in irradiated parts. (G.V.Goulyaev, 1971). Thanks to such an action ultra-violet rays influencing the molecule of DNA create new chemical links (N.P.Doubinin, 1970). Newly created links in the molecule of plastid DNA will lead to the changes of inherited information, and such a DNA molecule can be considered mutated, thus unable to react to the common changes of the environment with the former norm of reaction.
But in most cases the molecule of DNA can repair its initial structure. The plastids of plants that belong to Cactaceae are not exception.
The studies of the system of repairing ferments correcting the damage of genetic structures caused by irra-diation or chemical agents became a new milestone in molecular genetics. First repairing systems were found only at bacteria and protobe, but then it was proved that reparation also exists in fungus, algae and cells of superior plants (N.P.Doubinin, 1970). It is just for plastids too.
Among the best studied are two kinds of reparation - photoreactivation and dark repair. Photoreactivation is the reparation of DNA cells previously irradiated by ultra-violet rays and then lit by the visible light. The indispen-sable condition is the presence of photoreactivating ferment.
But it was found that in some cases you can also observe reactivation in the darkness. This kind of reactivation not connected with light quantum was called dark repair. Its molecular mechanism is absolutely differ-ent from photoreactivation. In case of dark repair both the damages caused by irradiation and chemical agents can repair. Repairing ferments of the system of dark repair "recognize" any changes in DNA breaking the Watson-Creak double spiral (the break of DNA secondary configuration), "cut them out" and then repair the native struc-ture with the help of repairing synthesis.
There are lots of other systems of reparation described. Among them we should mention thermal reactivation, reactivation with keeping in fluid and others.
When the intensity of ultra-violet rays increases chloroplasts and leucoplasts start synthesizing carotinoids. This is the defense reaction of plastids. The order to the synthesis is given by. In which way? The nucleic acids of plastids change at the intensity of influence different from optimal. Thanks to these changes plastids begin to syn-thesize other pigments. Then the nucleic acids of plastids repair thanks to decreasing of influence of lights of the certain spectrum. That means the changes are reversible.
The time for reparation of the changed plastid DNA differs at different groups of plants. It also differs at the plants of the same group kept in different conditions of having different intensity of nutrition. ("Experiments with fruit of Ariocarpus", Z.D.Semerenko). There takes place reparation of plastid DNA in initial cells of the shoot upon increasing of the intensity of nutrition thanks to implanting and also the reparation of plastid DNA in differ-entiated cells.
In the Nantaceae family there takes place another kind of reparation of plastid DNA. A would call this proc-ess meiotic reparation.
Low chlorophyll plants from many genera of Nantaceae family can produce seeds. But most of the seed-lings grown of these seeds are of normal green color. (F.Dorning, 1986). This is an example of reversible plastid mutation in the course of meiosis. What's going on? By different reasons some seedlings that are several days old have white, pink or yellow epidermis. Such seedlings exhaust the supply of nutrients from hypocotyl, and if not implanted in time they die. There are not many chloroplasts in such seedlings: not more than ? of the total number of plastids. The intensity of photosynthesis in such seedlings is very low. The general intensity of biosynthesis in such seedlings is very low which makes their development impossible.
Mutated plastids can not produce chlorophyll. Implanting is the only means to save such seedlings. In this case the stock synthesizes organic materials both for itself and for the cion.
If the care is appropriate the cion can bloom. In the course of meiosis gametal cells are produced. Cytoplasm of ovule together with plastids make the embryo of the seed. And then a miracle happens. The ripe seeds germinate and give 99% of green shoots. ("Chlorophyll free astrofitum" by V.Haage, O.Sadovsky).
Certainly it is the reparation of mutated DNA either in the course of meiosis or in the initial step of embry-onic period of ontogenesis. Anyway, plastid mutation at sexual reproduction of "colored forms" of cactuses is not inherited. But the above mentioned is right only for plastid mutations which did not appear under the influence of the cellular kernel.
The coloring of the epidermis of stems of some representatives of Nantaceae family is nonuniform. This feature in most cases is inherited. Gymnocalycium michanovichii and its varieties are an example of such feature.
Spots and stripes on the epidermis of the representatives of this species can be explained by the change in the balance of chromoplasts, chloroplasts and other groups of plastids in particular cells under the influence of cellular kernels. Apparently, the zygote produces cells with several different genotypes of kernels in the course of mitoses. So, the feature will be inherited according to Mendel's law.
Sometimes when you sow the seeds of Gymnocalycium mihanovichii var. Friedrichii you can find red or purple seedlings. The quantity of chloroplasts in the epidermic cells does not exceed 1/3 of all the plastids. The rest of plastids are mutated, it is impossible for them to synthesize chlorophyll. These mutations are the result of the influence of the cellular kernel. The karyogene (or a group of them) leading most of plastids to mutation can be in this case only recessive. The fact is that Gymnocalycium michanovichi and its varieties are cross-fertilized, het-erozygous and very changeable even within one population. Certainly the karyogenes leading plastids to mutation are of mutation origin.
For the germ of the seed to be low chlorophyll it should be generated from the zygote that is the result of the fusion of gametes giving only recessive gene. The possibility of forming such a gamete out of "wild seeds" is low enough. This possibility slightly increases in the collections where the plants with prevailing red epidermis were selected from generation to generation.
If the zygote is formed by the fusion of gametes of which one carries dominant genes and the other reces-sive the germ will be heterozygous, and its plastids will become red or green depending on the intensity of light, that is the plastids will not mutate or only a small part of them will. The above mentioned is right if the gametes of Gymnocalycium mihanovichii var. Friedrichii are haploid. If they are diploid or triploid the chance to form a ho-mozygous germ which is recessive concerning this gene is very low.
Sometimes the seeds of interspecific hybrids can give low-chlorophyll seedlings. That means there is a combination of certain genes in the kernel of zygote giving rise to plastid mutation. It is possible that such a cell synthesizes compounds preventing the synthesis of chlorophyll. In rear cases the descendants of interspecific hy-brids are low-chlorophyll. Among the examples there are hybrids Pseudolobivia kermesina x Echinopsis eyriesii and Pseudolo-bivia kermesina x Echinopsis eyriesii var. grandiflora.
Unfortunately I.Vatanabe in 1941 stopped cross-breeding and artificial selection and occupied himself with selection implantations. If you get the seeds of Gymnocalycium mihanovichii ?Hibotan?, which was raised by I.Vatanabe, by self-fertilization only a small part of them will give pink seedlings (not red). The other part will be green and brown with pink shades of different intensity. Though it is much easier to reproduce "colored" forms by implanting side shoots than by implanting 5-10 days old seedlings to temporary stock and then re-implanting them. But only implanting tiny low-chlorophyll seedlings, crossing them and selecting low-chlorophyll individuals re-peatedly sooner or later you can get stable recessive homozygous forms of, for example, Gymnocalycium mihano-vichii ?Hibotan?.
Having in mind morphological characteristics and discarding plants without clear divergence from green epidermis one can make dominant genes disappear without plastid mutation.
Such selection can be fulfilled not only with Gymnocalycium michanovichi but also with other representa-tives of the genus and with hybrids having clear divergence from green epidermis. The only condition for the se-lection is a divergence in the color of epidermis from green at the optimal environment at least at one of the parent plants. The above mentioned is also referred to the plants having the stem of different colors in the optimal condi-tions. By constant selecting individuals and crossing them one can achieve the maximum expressed and constant of the characteristic. But for getting two- or three-color plants most of authors use selection implanting. The resulted plants can preserve its "colored" characteristics only if reproduced by cloning.
One can get plastid mutation by using mutagens both of physical and chemical origin at germinating seeds, parent gametal cells and seedlings several days and even weeks old.
Even affecting germinating seeds by small concentrations of chemical mutagens and UV-rays one can achieve mutations in plastids DNA thanks to their high sensitivity to the outer influence.
Most of resulting plastid mutations is reversible independent of the way of affecting. But when cloning in most cases low-chlorophyll characteristic remains. Besides using mutagens is easier than selection implanting. "Colored" forms of cactuses can be got more quickly.
It is commonly known that if tiny seedlings are affected by sharp temperature pulldown the spatial length of plastid DNA helix will reduce. This is the way all the nucleic acids behave. But the reduced length of plastid DNA will remain at about a half of plastids of outer layers of cells of Astrophytum asterias var. nudum even after the low temperature influence. These plastids will stop producing chlorophyll. This is referred not only to the differen-tiated cells of epidermis but also to the cells of subepidermal layers and initial cells of apex of the stem of the seed-ling. The initial cells will split up and give rise to new low-chlorophyll cells. As a result the new outer cells will contain about half of the green plastids or may be less.
The place - the seed saucer or the implant - where the seedlings are exposed to temperature difference does not matter. The key is that this difference should be as sharp as possible and not less than 40° N and the seed-ling small enough (not more than 4 mm in diameter). Of course, this is a mistake to move a month old seedling of Astrophytum asterias var. nudum. from the greenhouse with +35° N to the refrigerator with -5° N. One should keep in mind the environment characteristic for the species in the wild nature. If the absolute minimum for Astro-phytum asterias var. nudum is about +8° C you should not lower the temperature more.
That is to say you should not place a "hot" seedling to the refrigerator with the temperature below 0 ° C. But low temperatures (down to -7° N) do not do much harm to Rebutia, Lobivia, Opuntia, most of representatives of the Chilean genera. The time of keeping seedlings in the refrigerator depends on its diameter and genius and is found by experiments. Each genius demands individual care. I believe that the time of "freezing" should not exceed 25 minutes. For example, a seed-ling of Astrophytum asterias var. nudum 4 mm in diameter was heat in the greenhouse up to 40° N and then moved to the refrigerator with 0° N where it was kept about 20 minutes. After that it was taken to a warm shady place and implanted into a Cereus peruvianus. In several days the bright-green seedling began getting paler. In about a fort-night the seedling started growing. Now its color was pale yellow-green which is not characteristic of this taxon at all.
Changing the temperature difference and the time of keeping seedlings in the refrigerator one can injure dif-ferent quantity of plastids, thus, changing the intensity of yellow and red coloring of the epidermis. The key is to influence the initial cells of apex of the stem of the seedling because these are they who are in charge of growing of outer cells layers.
There was a story that prompted me such an experiment. When my Melocactus peruvianus was about 5 years old I left it accidentally in the balcony in late September. I was in a trip and felt that it was getting colder. The next day I came back terrified by ice in the pools and frost on the grass. My balcony is on the first floor and it is open. There was no frost in the cactuses but the thermometer ran -2° N. For most cactuses 10 hours of staying at such a temperature will do no harm but Melocactus peruvianus is a heat-loving genius. In winter it was always kept at about +10° N. I took it to the room immediately.
In several days I found a transverse chain of small white spots on the side turned to the light. That was the result of unusual for Melocactus peruvianus low temperature.
Four years passed, but the spots still remain. The cause of their existence is clear. The plastids of outer dif-ferentiated cells were injured though the cells remained intact. The frost did not affect the initial cells as the plant was 6 cm in diameter.
There was no sharp temperature difference which saved most of plastids invariable.
The synthesis of chlorophyll depends on many conditions - plastid structure, different ferments of both cyto-plasm and plastid and so on. If any of these conditions is broken chlorophyll will not be able to produce.
If there is mutation of plastid DNA, theoretically two possibilities exist: either the plastid structure is violated or the synthesis of a ferment or a group of ferments stopped. Everything depends on the kind of plastid mutations. But anyway the changed plastid divides repeatedly and the number of mutated plastids inside the cell increases. As a result the mutated plastids go to the daughter cell.
I should say that it is difficult to get the mutation of the kernel DNA. These DNA can mutate only when the physical or chemical influence is very powerful. At least the above mentioned temperature difference will not give rise to the process. But plastids are very sensitive to the changes of the environment and can mutate at any un-common conditions. The key is to watch so that they should not adapt to these conditions.
I tried to treat the germinating seeds of different species of Astrophytum and their hybrids with UV rays twice as intensive as the ultra-violet radiation of the Sun in the mid-day. The irradiation was one-time and not very long (15 minutes). But for newly germinated seedlings it was very strong. The phenotypic reaction of the plastids followed in 2-3 days. The irradiated seedlings turned low-chlorophyll. The seeds that had not germinated by the moment of irradiation gave green seedlings. One of the Astrophytum senile var. Aureum seedlings turned bicolor - one edge is yellow, the rest are green. Apparently the seed skin defends the seedling of the ultra-violet influence. But if it is broken UV rays fall to it through the gap.
In this case the plastids of irradiated cells mutate and those not irradiated do not. It is a rare case but one can try to get bicolor seedlings this way. The key is to learn to scratch the skin in the right place.
It is a fact of common knowledge that during the period of replication the separating chains are especially sensitive to any mutogens and thus, affecting them during this period should increase the effectiveness of muta-tions. That means the best time for getting plastid mutations is during the process of vegetation when most plastids divide actively. Using UV rays one can consecutively denature DNA cells and at the same time affect the separat-ing chains with chemical mutagens. As experiments with inferior organisms proved chemical mutagens are espe-cially effective when combined with UV radiation (G.V.Goulyaev, 1971)
Plastids are certainly inferior organisms. If combined affection could give rise to irreversible plastid muta-tions many problems of growing "colored" cactuses would be solved. The species containing the most of irre-versibly mutated plastids could be reproduced both by vegetative and generative way. In both cases low chloro-phyll should remain. Such plants could be fertilized by the pollen of the plants with not mutated plastids and get low chlorophyll posterity whose flowers would be like those of paternal plant. The number of interspecific and even intergeneric hybrids is very large. The selection of colored forms would be very promising if it were not for vitally important for plastids repairing genes and DNA.
The test of plastid mutation for reversibility can take years. May be there is no sense in getting and testing such mutations? But I believe that such mutations are interesting both from the scientific and commercial point of view.
When trying to get low chlorophyll seedlings one should remember of plastid and kernel mutations as a re-sult of ageing of seeds.
In 1933 M.C.Navashin found the growth of frequency of mutations in the process of ageing of seeds of Gre-pus tectorum . The discovery was proved by the experiments held by A.Blacksly with Datura stramonium and oth-ers. In the experiments held by G.Shtubbe the frequency of recessive mutations of seeds of snapdragon kept during 5 to 10 years grew 14% compared to 1.5% of fresh seeds.
In the course of cytological research M.C.Navashin found that the frequency of chromosome anomalies es-pecially their breach increases as the germinating power of seeds decreases. The more intensive is the process of metabolism the faster is the ageing of seeds. It was proved directly by experiments. The seeds kept at the low tem-perature or in the conditions close to anaerobic at which their ageing slows down have less chromosome mutations in comparison with the seeds kept at the room temperature or in the conditions of normal access of air. The in-creasing number of mutations is connected with the growth of concentration of automutagens where lactic and acetic acids, aldehydes, alkaloids, coumarins and many derivatives of purines are referred. These are the substances which selectionists use for getting artificial mutations.
Editor's notes:so, according to the author, colored forms can be got at least by three methods:
- 1.overcooling of seedlings;
- 2. exposure to UV rays;
- 3. using old seeds
If you want to share your experience in gettig colored forms by other methods your articles are welcome.
Translation Irina Koudina, Mosсow, Russia, e-mail: firstname.lastname@example.org