Plant Hormones-CYTOKININS

 

Skoog and his students, while working on the callus cultures under in vitro conditions, Cytokinins were discovered.  They found the callus that develops from the stem explants, containing both pith and vascular elements, develops well.  But the explants containing just pith cells produces callus, but further growth of its stops, even in the presence of optimal concentration of auxins.  This is because the cell in the callus somehow rendered incapable of cell division.  If such callus is supplanted vascular tissues, extract of vascular tissues, coconut milk or malt extracts, the growth of the callus will be restored and the cells exhibit mitotic activity.  This effect has been attributed to the presence of some active principle in the supplanted coconut milk.  Cytokinins have various functions in association with other hormones or its accessories.

The active principle responsible for inducing cell division was isolated first from the extracts of yeasts.  Such a substance was called kinetin, later the name was changed and called as cytokinin; kinetin terminology was misleading for another class of compounds called kinins which were already known to be found is animal systems.  However, the term cytokinin has been given to all those compounds that are capable of inducing cell division in the presence of optimal concentration of auxin.   Now it is known that a good source for cytokinin is coconut liquid endosperm and milky endosperm of sweet corns.

 

Some of the naturally occurring cytokinins are 6-furfuryl-aminopurine, Ribosyl zeatin, Zeatin, Isopentinyladenine and Dihydrozeatin.  Interestingly, the above compounds are also found in denatured products of nucleic acids.  Many synthetic cytokinins are also available in the market, ex., 6 Benzyl aminopurine, 6 Phenyl aminopurine.

 

Distribution:

 

Cytokinins have been detected in a wide variety of plants; from unicellular yeasts, algae to multi cellular higher plants.  Particularly in higher plants, cytokinins are found in root tips, xylem, young leaves; endosperms of developing fruits, germinating seeds and tumour tissues.

 

Site of Synthesis:

 

Most of the cytokinins required for the plant body are synthesized in the root tips, and then they are translocated to different regions particularly to meristematic and expanding tissues; transportation is through xylem stream.  This observation has been supported by many studies. 

Site of CK synthesis

 

For example, the amount of cytokinin found in the excised petiole is less than the petiole that has rooted.  If the root tips are cut, the growth of the stem apex is more or less inhibited till adequate supply of cytokinin is restored by new root formation. Significant amount of cytokinin is synthesized in required endosperm of palm fruits, kernel of cereal grains and others.

 

Almost all naturally occurring cytokinins are the products of purine nucleotide derivatives.  The presence of iospentenyl adenine in some tRNAs has misled people to believe that the source of cytokinins is tRNAs but it is not the case.  Nevertheless the site of synthesis of cytokinins in young and developing plants is restricted to meristematic regions of the root tips; where certain enzymes utilize purine and convert them to cytokinins.  The presence of isopentenyl adenine in many tRNA is not due to the incorporation of cytokinins into tRNAs.  However, the exact mechanism and biosynthetic pathway of cytokinins is yet to be elucidated.

 

Effects: 

 

 

 

 

General Metabolism:

 

Cytokinins’ effect on respiration is very interesting.  It enhances the rate of respiration in the callus, but the same hormone when applied to senescing leaves, brings down the rate of respiration, retards the degradation of chlorophyll and enhances the rate of chlorophyll synthesis.  In addition it also increases the rate of metabolic activities involved in C3 pathway.  In certain systems cytokinin induces nitrate reductase activity, but in callus it does not.  The root nodule development in legumes and its metabolic activity is subtly regulated by the interaction between auxins and cytokinins.

 

Nucleic acid Synthesis:

 

Although cytokinin is known to stimulate cell division, it does not induce DNA synthesis as a prelude to cell division.  But in the presence of auxin, it promotes DNA synthesis.  So it is suggested that cytokinin stimulates and auxin promotes DNA synthesis.  For example, an aged and non proliferating callus cells can be stimulated to undergo mitotic divisions by the application of cytokinin.

 

Cytokinin binding protein i.e. Receptor proteins have been identified in many plant systems.  The cytokinin protein complex is known to induce transcription activity a pea bud chromatin in a cell free system.  In Soy bean and French been hypocotyls, it transitorily inhibits IBA induced RNA synthesis for a period of 6-7 hours, but later it stimulates RNA synthesis.

 

Specific species of tRNAs, containing cytokinin activity, is seen in the cotyledons and hypocotyl segments of bean plants.  But this has been identified as due to post transcriptional modification of tRNA.  Recent investigations have clearly elucidated that the presence of cytokinin moiety in some tRNA structures is not responsible for eliciting any cytokinin mediated responses, however it is to be noted that many tRNAs contain isopentenyl adenine (IPA) in the anticodon loop region

 

PROTEIN SYNTHESIS:

 

Quite a number of experiments, involvement in vivo systems or cell free invitro systems, have demonstrated that cytokinins first stimulate translational activity without any concomitant increase in the synthesis of mRNAs.  This particular effect has been attributed to cytokinin’s ability to activate pre existing mRNPs and ribosomal proteins needed for chain initiation. Fox et al, have demonstrated that cytokinins bind to ribosomal surface through a receptor protein.  This protein activates ribosomes by dephosphorylating a specific ribosomal protein.

 

Furthermore, the increased activity of protein synthesis in response to cytokinin has been found to be concentration dependent.  It is also interesting to note that cytokinin mediated protein synthesis; either as short term or long term effects, shows the synthesis of new proteins in treated tissues.  At least in the hypocotyls of French bean, cytokinin inhibits some proteins that are induced by IBA, but cytokinin by itself enhances the rate of tubulin synthesis similar to that of IBA.

 

Cell Division:

 

The name cytokinin is derived from its ability to induce cytokinesis during cell divisions. Though cytokinins stimulate the process, the permissive effect of it is controlled by auxins. When cytokinin is provided to a liquid culture medium containing plant cells, the protein synthetic activity of the cells is greatly stimulated.  Moreover, some of the proteins thus synthesized are new ones.  These observations suggest that cytokinins control cytokinesis by regulating the synthesis of some specific protein factors that are required for cytokinesis.

 

Cell Enlargement:

 

Isolated cotyledons of Cucumis, radish and other plants expand dramatically when they are treated with cytokinin.  The expansion of cotyledons is rather more due to cell enlargement than due to cell divisions.   During cytokinin induced cell enlargement, respiratory activity increases significantly and greater amounts of K+ ions are accumulated in the cells.  At the same time, cells in response to the hormones induce the synthesis of few minor species of RNAs and some proteins.  But the inhibitors of respiration, transcription and translation completely inhibit cytokinin mediated cell enlargement.  Interestingly such cotyledons also respond to red light treatment and enlarge.  However the red light induced enlargement cannot be reversed by far red light treatment which further suggests that cytokinins bring about permeability changes within the membranes.

 

 

Richmond & Lang’s effect:

 

Senescense of leaves leads to yellowing and finally leads to the fall from the plant.  If a young excised leaf is kept in water, it slowly changes its color to yellow and dies.  If such leaves are provided with cytokinin, the yellowing is significantly delayed and such an effect is called Richmond and Lang’s effect; named after the discoverers.

 

Senescense is a common feature exhibited by parts of the plant which show definite growth pattern.  With age, the structures like leaves, flowers, etc., senesce and die.  Among various factors, decrease n the content of auxin acts as a very important factor in inducing senescence.  In matured leaves, still attached to the plant body with time, senescence sets in and leads to the degradation of chlorophyll.  Catabolic activity increases and the formation of abscission layer begin.  In detached leaves also one finds similar processes.  But cytokinins prevent and prolong the initiation of senescence for a quite a period of time.  This effect of cytokinin has been explained as due to the prevention of degradative catabolic processes by the way of repression activity of few hydrolysing enzymes like protease, RNAse, DNAse etc.  Furthermore, cytokinin facilitates the chlorophyll synthesis.  It also sustains the activity of carbon fixation, RNA synthesis and protein synthesis.  Still the exact mechanism by which cytokinins prevent aging and senescence is not known.

 

Effect on Dormancy:

 

Dormant buds that develop due to certain adverse environmental factors remain inactive for a long time.  If such buds are treated with cytokinins they come out of dormant state and sprout.  This is due to the effect of cytokinin in activating cell division which was prevented by mitotic blocks present in the dormant besides.  Interestingly, cytokinins also overcome auxin imposed apical dominance and stimulate the growth of the axillary buds, probably by overcoming the factors such as mitotic blocks.

 

Interaction of cytokinins with auxins in morphogenesis:

 

Plant development is a series of biochemical events which ultimately leads to morphogenic changes.  This process is regulated by a number of phytohormones which bring about differential gene activity leading to the development or specific phenotype.  The interaction between various hormones is very complex and they operate at different levels like differentials transcription, protein synthesis, enzyme activity, permeability etc.  Here the interaction between cytokinin and auxin id discussed briefly.

 

The culturing of plant explants with various combinations of cytokinins and Auxins show the inhibitory effect of cytokinin on auxin induced new root formation.

 

In tissue culture experiments, the plant explants are supplemented with optimal nutrients including carbohydrate as the energy source and hormones as growth regulators.  Plant explants, which may be leaf segments stem segments, roots or embryos develop and produce an undifferentiated tissue called callus is a defined culture medium.  This behavior is due to the presence of balanced concentration of auxin and cytokinin in the nutrient medium.  The callus is like a cancerous tumour, where the cells are undifferentiated and they are under the spell of uncontrolled mitotic activity.  If the concentration of auxin with respect to that of cytokinin ratio is changed, from the ratio that is favorable for the growth of only callus, the cells of the callus undergo transformation to produce organs like roots or shoots.  If the ratio between auxin and cytokinin is high, the callus cells undergo transformation and produce roots.  On the other hand, if the ratio between auxin and cytokinin is low the callus cells initiate shoots.  As all cells in the callus are totipotent, depending upon the hormonal concentration, cellular genetic material is differentially expressed and their products induce specific morphogenetic events leading to organ formation.  In this system it is the concentration of each of the hormones provide the signal for differentiation of undifferentiated cells into new organs.

 

In spite of having sufficient knowledge about the hormonal effects in organogenesis, the molecular basis of morphogenesis is not very clear; however, one example can be used to illustrate how auxins and cytokinins interact and modulate molecular events during morphogenesis.  Adventitious root formation in the hypocotyls of phaseolus vulgaris in response to auxin treatment is a process of redifferentiation and reorganization of pericyclic cells into root primordia.  While IBA induces new root formation, cytokinins inhibit IBA mediated root initiation.  At molecular level both auxin and cytokinins increase the rate of protein synthesis without increase in transcriptional activity at 30 minutes of treatment.  In the case of auxin treated hypocotyls increase in transcription is detected after 45-60 minutes, but not in cytokinins treated.  In the segment treated with both auxin and cytokinins one does not observe such changes at short time but observed at longer periods.  This observation comes from quantitating in vivo and in vitro protein synthesis using 14C leucine.  Also purified poly-A is used for quantitation using in vitro translation.  The results show increase in translation at later stages is due to transcriptional activation. General Protein analysis on SDS-PAGE at 24hrs, 48hr and 72 hrs, shows a remarkable increase in two bands at 55Kda and 58Kda and few other bands of high molecular weight.  However increase in the said proteins was not detected in cytokinin treated segments.   The bands were identified as Tubulin alpha and beta proteins.  More over in Auxin treated segment discerned from in vitro translation of Pol(A) RNA shows one more 115Kds band showed up only in auxin treated but not in any others.  This increase is only transitory from 24hrs to 36hrs.  Then the protein band disappears.  The role of this protein is not known.

At molecular level Auxin acts through Auxin response factor for activation of genes.  But the activation of transcription at 36 hrs or more, by cytokinins is yet to be found.  However cytokinins action of activation of genes has been discerned to some extent in Arabidopsis.  Cytokinin acts as signal molecule and it binds to dimeric receptors anchored in plasma membrane.  The receptors are believed to be similar to receptor tyrosine kinases (RTKs), but their kinase activity  is restricted to histidine residues (HPK), so they are called Arabidopsis histidine protein kinases (HKs).  This kinase activates histidine phospho transfer proteins (AHPs) transducers.  These are response regulators which can activate or repress gene expression.  AHK-ps enter the nucleus and interact with ARRs (nuclear response regulators) and activate transcription. Such components were also observed in maize.

Outline of cytokinins pathway

There are four steps in cytokinins signaling pathway as shown in the obove diagram. AHK sensing and signaling, AHP nuclear translocation and localization and activation of ARR genes and a negative feedback loop through Cytokinin inducible ARR gene products.

Arabidopsis encode three cytokinins receptors; cytokine response gene1 (CRE1 also called AHK4) ,Woodenleg (Wol) and AHK2/3.  There are other histidine kinases such as CK11 and CK12 (AHK5) which are independent of cytokinins but they do respond to cytokinins.  Mutants of CRE1 and Wol show defects in cytokinins mediated shoot formation and defects in root vasculature.

The ARR (A response regulator proteins) ( such asARR1, ARR2,and ARR10, which are transcriptional activators carry MYB like DNA binding domains, and also contain glutamine Q’ rich activating domains.  ARR p-lation activates transcription of A-type ARRs.  Expression of cyclin D and ARR5 show they are the major sites of cytokinins action in root and shoot meristems.  There are 54 gene encode AHKs, AHPs and ARRs.

 

 

 

 

 

 

Cytokinins requirement for nodulation

 

 

 

Signals that control plant vascular cell differentiation

 

 

 

 

 

In procambial cells (PCs), the coordinated signaling by cytokinin and auxin induces the expression of genes that are involved in the maintenance and growth of procambial activities into xylem elements. The auxin-signaling pathway might be involved in gene expression of auxin-response factors, such as MONOPTEROS (MP), that also function as transcriptional activators, and their repressors, the AUX/IAA proteins. Cytokinin might be perceived by the WOL/CRE1/AtHK4 cytokinin receptor, which, in turn, transmits an intracellular signal that is mediated by a His–Asp phosphorelay mechanism to PC-related histidine-containing phosphotransfer factors (AHPs) and then to PC-related type-B response regulators (ARRs). The type-B ARRs might function as transcriptional activators of PC-related genes including the genes of their repressors, the type-A ARRs. The presence of repressors in auxin- and cytokinin-signaling pathways might allow cytokinin and auxin signaling to be temporal. Brassinosteroids (BRs in the figure) are biosynthesized actively in PCs and secreted, but brassinosteroids do not work as a signal for the maintenance of procambial activities. Instead, brassinosteroids, in the presence of auxin, might initiate differentiation of procambial cells to precursors of xylem cells (pXCs) after recognition by a receptor, which might be a heterodimers, composed of either brassinosteroid-insensitive-1 (BRI1) or one of the BRI1-like proteins (BRL1–BRL3), plus BRI1-associated receptor kinase-1 (BAK1). The brassinosteroid signal inactivates the negative regulator BIN2 (brassinosteroid-insensitive-2), which allows the unphosphorylated form of bri1-EMS-suppressor-1 (BES1) and brassinazole-resistant-1 (BZR1) to translocate to the nucleus and to promote pXC-related gene expression. Among the most important pXC-related genes that are induced by brassinosteroids might be the HD-ZIP-III-homeobox gene family, which might function in further xylem cell differentiation. KANADI and the microRNAs MIR165 and MIR166 might suppress differentiation of PCs to pXCs. The suppression by the microRNAs might be caused by the rapid degradation of the HD-ZIP-III gene mRNA through RNAi machinery.

The above diagram shows how cytokinins play a role in localization of CRF6 in cytosol.

 

 

RFs in Arabidopsis

 We have taken a genetic approach identifying knockout mutants in each of the CRF genes in order to better understand their function in plant growth and development.
One research area we on is determining the b

 

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Cytokinins induce CRF genes.  There are six such CRFs; they are cytokinin response/regulatory factors and transcriptional activators.  They play important role in plant development. Mutants in CRF genes produce malfunctions in leaf and cotyledon development as shown in the diagrams above. 

 

This figure provides general information about cytokinin signal pathways. Arabidopsis, each of which into pairs of related genes that arose as ancient genome duplication in plants. Each pair of genes has been maintained over time and may have become specified in function.
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While only three have taken a genetic approach identifying knockout mutants in each of the CRF genes in order to better understand their function in plant growth and dFig. 4.

Cytokinin activated gene expression is shown in the form of microarray.

CRFs act in parallel with type-B ARRs to mediate cytokinin regulated gene expression. (A) Wild-type, arr1,12, crf1,2,5, and crf2,3,6 seedlings were treated with either 10 μM BA or a DMSO control for 1 h and gene expression analyzed by using a microarray. Genes that displayed a ≥2-fold change in response to cytokinin in the wild type are shown. (B) Venn diagram of the 135 cytokinin-regulated genes affected by the arr1,12, crf1,2,5, and/or crf2,3,6 mutations. (C) Model of cytokinin signaling. Both AHPs and CRFs move into the nucleus in response to cytokinin. Once there, the AHPs phosphorylate the type-B ARRs, which, together with CRFs, mediate cytokinin-regulated gene expression.

Another diagram shows few more Cytokinin signaling features.