Physiology of VERNALIZATION

 

VERNALIZATION AND FLOWERING

 

Light has a profound influence on plants and it performs many important biological processes like photosynthesis, phototropism, photorespiration, photoperiodism, etc.  Influence of light on plants in flowering is very fascinating, but not all the plants respond and flower to photoperiodic treatments.  Similar to light, the temperature also has significant effects on plant growth, dormancy and flowering.  Most of the metabolic processes are regulated by the temperature that is prevailing in the environment.  But the effect of temperature in inducing the development of reproductive organs is fascinating.

 

As mentioned earlier, different plants have different cycles of vegetative growth, flowering and fruiting.  However, some biennials which produce vegetative structures in one season and induce flowering in the flowering season only after they are exposed to prolonged winter or cold treatment.  Interestingly, such cold requiring plants also need proper photoperiodic treatment for flowerng.  Such biennial plants can be made to flower in the same season by subjecting them to cold treatment.  Hence the process of acquiring the ability or capacity to accelerate the process of flowering, in response to cold treatment is referred to as Vernalization.  In Soviet Union, the same phenomenon is called Yerrovization or Lysenkoism.  Ever since Gassner (USA) discovered this unique phenomenon, a large number of plants which required vernalization have been identified and detailed studies have been made about the behavior of such plants.  After the second world war, under the supervision of Lysenkov in USSR.  Extensive work has been done on vernalization in USSR and the same was exploited in the field of agriculture.

 

The winter rye called Secale cereale or Petkus rye is a biennial plant which requires cold treatment for successful cultivation as one season crop.  The grains of these plants are known for their hardiness and quality for the purpose of milling and baking.

 

In fact, farmers used to cultivate this variety of subjecting the water imbibed grains to cold treatment and growing them in the spring and harvesting in the same in summer.  After Klippert, who reported this phenomenon, extensive research work has been done in this field.  Among many plants, Petkus rye (short day plants) was the first to be used for experimentation.  The other examples are Hyoscyamus niger (long day plant) Triticum aestivatum (CV winter wheat), Lunaria bienensis, Arabidopsis thalliana, Lolium  perennial, Beta Vulgaris, Brassica oleracea, etc.

 

Age and Site of Vernalization:

 

Vernalization through cold treatment is very effective at the seed stage or seedling stage.  In some cereals, even the embroyes can be successfully verbalized. However, in many cold recurring species, vernalization is not effective until and unless the plant possess at least few leaves.  The requirement of few leaves for effective vernalization is called ‘Ripeness to Flowering’.  This suggests that plants need certain degree of photosynthetic obtain to respond for cold treatment.  Additional support to this view comes from an experiment on the embroyes of petkus rye.  The embryos are separated from the endosperm and then if the embryo alone is subjected to cold treatment, embryos fail to be verbalized.  But if such embryos are subjected to cold treatment along with carbohydrates like sucrose solution or endospermous tissue, vernalization is very effective.  Probably the role of carbohydrates in vernalization is to supply some energy.   Nevertheless, the most sensitive site which acts as the perceptive organ is the meristematic region of the shoot apex.  Even leaves which act as the sites should have certain amount of meristematic tissues in them.  But how does the cold treatment brings about this effect in such active cells is not known.

 

Temperature Effect:

 

For the normal growth and development, every plant requires on optimum temperature.  But for vernalization the optimum temperature required is 3 0C to 17 0C, which varies depending upon the species involved.  Even the duration of treatment varies from species to species.  Individual requirements have to be determined independently by experimentation.  In petkus rye the most effective range of temperature is 3 0C to 7 0C.  But for Hyoscyamus niger 3-17 degree is optimal.  However the efficiency of cold treatment in bringing about vernalization is determined by the number of days shortened between germination and flowering stage.

 

Effect of water and oxygen:

 

By just treating the seedling with cold temperature, the said structures do not get verbalized.  Along with the cold treatment plants also require water and oxygen for effective vernalization.  The seeds or embryos should possess at least 40-50% water in their cells, without which cold treatment has no effect.  Similarly oxygen is very essential; probably it is required for biological oxidation.  The essentiality of carbohydrates for effective vernalization supports the view of requirement of oxygen.  Still, it is difficult to explain how cells use carbohydrates and oxygen for enzymatic oxidative process at such low temperature.

 

Vernalin:

 

Various experiments are the past have revealed that during cold treatment, the meristematic cells found either in stem apex or leaves are stimulated to produce some substance.  The presence of such substance has been demonstrated by grafting a vernalized plant to another non vernalized plant at normal temperatures.  The plant that receives the graft, after sometime, starts producing flowers, which suggests some substance found in the vernalized plants is transplanted to non vernalized plant and that is responsible for the induction of flowering in the latter plant.  In some cases, if the cut shoot tip of the vernalized plant is placed above the decapitated stem of the non vernalized plant, flowering is induced in the receiver plant The above experiments clearly demonstrate that some substance is synthesized and such substance is now called ‘Vernalin” and it is capable of diffusion.  Attempts to isolate and identify the components of vernalin have failed.  Whether the vernalin is the same as florigin or a precursor of florigin is not known.

 

Devernalization:

 

If vernalized seedlings or seeds are subjected to higher temperature like 35-40 0C the plants that develop from such treatment fail to flowers.  Such a nullifying effect by higher temperatures is called Devernalization.  Nevertheless, if the vernalized plants are maintained at sufficiently low temperatures for a long period of time, which has to be determined for every species, devernalization is not possible.  This may be due to the putative vernalin have already acted upon the genetic material and committed it is flower formation.  However, devernalized plants can be revernalized by subjecting the same seedling or seed again for another period of cold treatment by repetition of vernalization and devernalization cycles.  Prolonged vernalization the effect decreases and seedlings loose their viability and potentiality to produce flowers.

The effects of high temperature interruption during low temperature treatment on inflorescence formation in turnip plants (Brassica rapa L. cv. Hikari) were investigated. Plants were exposed to 9 (low temperature treatment) with interruptions of 17, 24 or 30 for 4, 6 or 8 hr per day. After the treatment, flower formation indices (%) were calculated. Based on these data, the total hours of vernalization, and hours of vernalization or devernalization per day were estimated. Flower formation indices were lower in the group of plants exposed to high temperature interruption than in the group of plants exposed to continuous low temperature. Moreover, the higher the interruptive temperature became and the longer its duration per day, the lower the flower formation index became. The indices were particularly low, when turnip plants were exposed to temperatures of 24 or 30 for 6 or 8 hr per day. The influence of high temperature interruption during the low temperature treatment was determined quantitatively by measuring the estimated hours of vernalization and devernalization per day. For example, vernalization effect of 9 was diminished by 50 %, when the plants were exposed to 17 for 8 hr per day during the low temperature treatment or to 24 or 30 for 4 hr per day during the low temperature treatment. Moreover, the vernalization effect of low temperature treatment was diminished by 90 % when plants were exposed to 24 or 30 for 8 hr per day during the 9 treatment. This can be called as Devernalization.

 

Gibberellin as substitute for vernalization:

 

Many plants which require cold treatment also require proper photoperiodic treatment for the induction of flowers, without which vernalization does not have any effect.  If such plants are treated with gibberellins, they produce flowers without subjecting the plants to cold and photoperiodic treatments.  For example, Henbane is a rosette leaved long day plant which requires cold treatment for flowering.  If such untreated plants are sprayed with GAs, the plants produce flowers. It means gibberellins not only substitute vernalization but also photoperiodic treatment.  But same gibberellins have no effect on other long day and cold requiring plants species called petkus rye.  In some cases for the proper response to GA treatment, the plant should possess a cluster of leaves in rosette form as a precondition.  The effect of GA on plants like Henbane has been attributed to its effect on the elongation of internodes at which time GA also promotes and probably elaborates the factors required for the induction flowers.

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Cold treatment activates VER203 gene expression.  GA also induces VER203 expression thus GA can partially substitute cold treatment to initiate flowering in plants that require cold treatment.

Mechanism of floral induction in vernalized plants:

 

It is clear from the earlier discussions that the plants with their specific genetic make up respond to different treatments like cold or photoperiods and produce flowers.  Most of the cold requiring plants also require proper photoperiodic treatment.  Gibberellins are known to overcome both cold treatment and photoperiodic treatment in long day plants, but it has no effect on short day plants.  Synthesis of some unknown substance called vernalin during the period vernalization has been clearly demonstrated by grafting experiments.  Furthermore for proper vernalization, plants require sufficient amount of water, oxygen and some vegetation growth.  Though all the above said factors are provided to the plant, flower inducing substance won’t be synthesized until and unless it is treated with proper cold condition at the stage of its development.  It is during the cold treatment, the synthesis of the said flowering inducing factor is believed to be accelerated.

Winter annual requires cold treatment for flowering but Rapid recycler does not require veranlization

 

According to the hypothesis proposed by Purvis et.al. (older view), plants normally synthesize a substance called ‘B’ from some unknown precursor called ‘A’.  The synthesis of B is accelerated by cold treatment, when  ‘B’ is synthesized; it persists for a period of time.  Further conversion of B to C and then D is under the control of photoperiods.  The substance D is supposed to be the flowering hormone and C an intermediate compound.  The conversion of B to C is slow, but once it is formed it is rapidly converted to D.  Once D accumulates to a critical level, it induces flowering.  If the same verbalized plant is kept under short day conditions the conversion of B to C is inhibited, instead C is converted to B and then B is converted to E. So the plant remains in vegetative conditions. In fact today scientists have shown that cold treatment induces the expression of CO which in turn induces induces FT, which with  FD induce the expression of floral meristem identity genes.  These on expression act on floral part inducing genes.

 

This particular hypothesis is based on the studies on perks rye.  This plant even if it is not verbalized  and grown under normal temperature and it proper photoperiodic treatment is given, it still flowers, but it takes a long time.  On the contrary, if it is subjected to vernalization and then subjected to long photoperiod, it produces flowers in a shorter time.  It means that the synthesis of A to B takes place all the time, but it is accelerated under cold treatment.  Once B accumulates in sufficient amounts, B is converted to C and then to D, which is actually under the control of long day conditions.  But in short day or day neutral conditions C is converted back to B and then to E which keeps the plant in vegetative conditions.

 

Though this hypothesis appears to explain some observed phenomenon in petkus rye a long day plant, it does not offer any explanation how short day plants flower what is the nature of the intermediate compounds.  There are more ambiguities in this theory, than it explains.

 

Chailkhyan, on the other hand is of the view that floral induction requires two substances.  One is GA or GA like compound and the other is flower inducing substance.  So he assumed that cold requiring long day plants contain enough flowering substance but lack in GA like compound which will be synthesized under inductive conditions.  Contrary to this, cold requiring short day plants possess sufficient amount of GA like compounds and lack in flowering substances which will be synthesized during inductive conditions.  The GA like compound and flowering substance combine to produce a flower inducing complex called florigin.  Whether it is a cold requiring plant or photoperiod requiring plant or a plant which requires both, the hormone that is required for flowering has to be the same.  Chailkhgan’s view was supported by G. Melcher, who grafted a short day Maryland mammoth plant (it does not require cold treatment), to another non–verbalized cold requiring long day Hyoscyamus plant.  Grafting resulted in the formation of flowers in Hyoscyamus plant even under non inductive conditions.  .

The explanation is that each of these plants possesses one of the two essential substances and lacked the other, i.e., short day plant contains GA and the long day plant possesses the flowering substance.  Due to grafting the GA from short day plant diffuses into long day plant, where both combine to produce a flower inducing substance or florigin, which inturn induces.  In spite of numerous efforts to isolate and identify the vernalin or the flowering hormone, have remained elusive.  Presence of some such substances has been established by experiments but the nature of that material is not known.  More than that, nothing is known about the molecular events that lead to flower formation in response to cold treatment.  Whether cold treatment induces any gene expression or whether cold treatment destroys some repressor proteins that are bound to MRNAs there by activities pre-existing MRNPs, whether vernalization brings about the destruction of a repressor, there by activities a gene expression are few of the questions for which there is no answer.  But one thing is certain that during vernalization period some thermo labile component is produced and it remains for a long period of time in the plant and it works with another substance produced during photoperiodic conditions.

Actually plants requiring vernalization contain FLC protein bound to specific  loci that represses genes required for flowering.  It is during cold treatment the FLC dissociates from the loci and allow other factors to bind and activate gene expression.  It is this that makes the plants to flower in response to vernalization.

 

During cold treatment, with time the concentration of FLC, the repressor goes down, means its binding to chromatin gets reduced with time under cold treatment.

 

 

 

 

 

 

 

 

Arabidopsis as a model system is used to study the regulation of flowering time. In addition to the general advantages (small sized, rapid generation time, sequenced genome, and a large collection of gene knock-out lines), flowering in Arabidopsis is regulated by both photoperiod (flowering is accelerated by long days) and vernalization. Early-flowering lab strains of Arabidopsis do not have a vernalization requirement for early flowering; however, naturally occurring winter-annual accessions of Arabidopsis are late flowering and vernalization responsive due to dominant alleles of the FRIGIDA (FRI) gene. A number of late-flowering vernalization-responsive mutants (such as FLD) have also been isolated from mutagenized early flowering strains. The genes identified by these mutants are collective known as the autonomous pathway floral promotion pathway.

 

 

 

 

FLOWERING LOCUS C (FLC) blocks flowering prior to vernalization

Work has shown that elevated levels of the MADS-domain-containing transcription factor FLC cause the late-flowering vernalization-responsive phenotype of naturally occurring strains containing FRI or autonomous pathway mutants. FLC acts to block flowering and FRI, in turn, acts to up regulate FLC levels. The autonomous-pathway genes promote flowering by repressing FLC expression; thus autonomous pathway mutants contain elevated levels of FLC and are late flowering.

 

 

 

Vernalization promotes flowering by causing an epigenetic shut off of FLC

Our work has also demonstrated that the promotion of flowering by vernalization is also regulated by FLC. The late flowering phenotype of FRI or autonomous pathway mutants can be effectively suppressed by vernalization. This suppression takes place at the level of FLC regulation. Following vernalization FLC expression is permanently downregulated; for the rest of the plant's life FLC levels remain suppressed. Levels are then reset during reproduction and are high again in the next generation.

Vernalization promotes flowering by causing an epigenetic shut off of FLC

 

 

 

 

 

 

 

 Work has also demonstrated that the promotion of flowering by vernalization is also regulated by FLC. The late flowering phenotype of FRI or autonomous pathway mutants can be effectively suppressed by vernalization. This suppression takes place at the level of FLC regulation. Following vernalization FLC expression is permanently downregulated; for the rest of the plant's life FLC levels remain suppressed. Levels are then reset during reproduction and are high again in the next generation.

 

 

 

 

 

Silencing or reducing the concentration of VRN during cold treatment is shown, where VEL1, 2 and 3 bind to VRN at C-locus to offset effect of chromatin silencing in the locus.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

We a currently working to understand in greater detail how FLC is regulated by FRI, the autonomous pathway, and vernalization. We used a variety of genetic and molecular approaches in this pursuit. Recent work by our lab and others has shown that changes in chromatin structure are critical in FLC regulation. One gene recently cloned by our lab, EFS, is required for high levels of FLC expression; efs mutants suppress the late-flowering effects of FRI and autonomous-pathway mutants. EFS encodes a SET-domian containing transcription factor. SET domain proteins are known to act as histone methyl transferases, which can modify chromatin structure and thereby regulate gene expression.

 

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