In this article we will discuss about the transport of growth hormones in plants.
Non-Hormonal Factors:
Carbon dioxide has been known for a long time to stimulate plant growth. There are at least two CO2 responses which depend on the CO2 concentrations. One is the responses to low concentrations, e.g. 0.03% and the other to much higher concentrations. The characteristics of the two responses are different.
It is not clear whether CO2-stimulation is an acid effect or is a separate effect. Some workers like Hardy and Quebedeaux have designated oxygen and CO2 as key growth regulators. The concentration of oxygen in a plant regulates total growth of most crops and seed growth for all tested crops.
Obviously an extension of thought beyond the traditional plant hormones has been made by the suggestions that the two gases are key growth of roots and shoots of soybeans is dramatically improved in plants grown at 5% atmospheric oxygen, while pod and seed development on the same plants is greatly suppressed.
Carbon dioxide when increased to 800-1200 ppm has increased almost six- fold they symbiotic biological nitrogen fixation of soybeans.
How do Hormones Function?
The sequence of events involved in hormone function comprises two stages: the initial signal perception, and signal transduction pathway and final response.
Signal perception:
It is the reaction of the hormone with a receptor site. Hormones pass from cell to cell through diffusion, through apoplastic space or through plasmodesmata (Fig. 20-15). The responding cell is termed as target cell. The latter must have potential to decipher the presence of the hormone molecule and accomplish interactions between hormone and a cellular receptor which is specific of the target cell and is also specific for the hormone.
The receptors are glycoproteins which bind to the hormone and hence its conformation is altered and assumes an ‘active’ state. This active hormone- receptor complex leads to the generation of a signal. The specificity of the receptor determines which hormone will form the complex and attain the status of responding (Fig. 20-16).
Different cell types may have different receptors that in turn elicit different responses to the same hormone. This also shows multiple effects caused by a hormone, or several hormones causing the same effect.
This is followed by the signal transduction and amplification stage.
In this a set of biochemical events are set in motion in a cascade fashion leading to a final response. It is assumed that hormone-receptor complex activates a membrane protein, G-Protein which then binds to a third membrane protein e.g. adenylate cyclase located in the membrane surface (e.g. in case of insulin and epinephnerine in animals).
This binding activates the enzyme and stimulates the synthesis of cAMP in the cytoplasm. G-protein may also interact with an ion channel that regualates the flow of calcium in the cell. The Ca2+ may bind to calmodulin and one or the both activate specific protein kinases (Fig. 20-17).
The hormone is regarded as a first messenger since it carried the original ‘message’ to the cell surface. cAMP and calcium serve as second messengers. The latter relay the information from the plasma membrane to the biochemical machinery within the cell. Second messenger also amplifies the first or original message.
The above description holds for the animal hormone. Let us consider whether plant hormone action has any homology or analogy with it. Three aspects need to be considered and these are: do plants have hormone-binding proteins: do plants have second messengers, and do plant hormones change gene action?
There are some suggestive criteria to define proteins which bind with the hormones and these are: specificity of binding i.e. it should bind with a type of hormone and its structural analog. Second, the receptor should show a high affinity for the hormone. Third, if the concentration of the hormone is enhance it should saturate the receptor. Fourth, the hormone should bind reversibly with the intended specific receptor.
Auxin-Binding Proteins:
These proteins have been identified in callus culture and maize coleoptiles. In tobacoo callus tissue, three classes of IAA- binding proteins have been identified and two are associated with membrane functions. Some workers have suggested that the cytoplasmic-nuclear auxin-binding proteins are probably receptors.
In maize coleoptile, only membrane-associated auxin-binding proteins have been characterized. Using antibodies, against the auxin binding proteins, these were localized in the outer epidermal cells. It is proposed that the binding proteins are involved in auxin-induced coleoptile elongation.
Cytokinin-Binding Proteins:
Both soluble and particulate binding sites have been reported for this hormone and most extensively identified protein is CBF-1 protein from wheatgerm and appears to be loosely associated with ribosomes fraction. This protein appears to be associated with protein biosynthesis, as proposed one of the functions of the cytokinins.
Gibberellin and ABA-Binding Proteins:
For gibberellins no specific reports on the GA-binding proteins is available. The same situation prevails for ABA and ethylene. However, in guard cells ABA-binding site on cell protoplast is indicated and these sites appear to be proteinaceous being located on the apoplastic surface of the plasma membrane.
The overall conclusion is that direct evidence for hormone receptors in plants is still obscure.
Second Messengers in Plants:
cAMP has been discovered in plants as well but plant responses sensitive to cAMP are still obscure. Thus, most evidences do not support the second messenger role of a cAMP in plants. However, other molecules are suggested as second messengers in plants and these are calcium and phosphoinositides.
Calcium is reported to regulate large number of processes in plants e.g cell elongation and division protoplasmic streaming, activity of various enzymes, affect calcium-binding proteins-calmodulin (Fig. 20-17).
These proteins have been isolated from diverse plant species, and are appreciably similar to those isolated from animals. It may be stated that calcium would only function as second messenger if the cytosolic concentrations remain low and under metabolic control.
Cacium is stored in ER, mitochondria, vacuole and calcium-ATP ases activity regulated their level in the cytosol. Light, hormones mediate such a concentration. Calcium binds with calmodulin to form CaM. Ca2+ and thus activates various enzymes. Kinases and protein kinases are stimulated by this complex.
In plants there is good evidence to support the existence a calcium-mediated signal transduction pathways. Several questions regarding stimulus-response remain to be answered. The demonstration of calcium channels which are hormone mediated and present in the plasma membrane still remain to be demonstrated.
Phosphoinositides:
Inostitol triphosphate system appears to operate in plants (Fig. 20-18). The view is that phospholiphase C which is membrane enzyme is activated by the hormone receptor-complex and then acts through G ptotein. Phospholipase C catalyzes the breakdown of PIP2 to IP3 and DAG and the latter two substances function as second messengers.
IP3 moves in the cytoplasm where it stimulates the release of Ca2+ from the vacuole and DAG activates protein kinase C most of these studies remain to be undertaken in details. Several other second messengers are suggested in plants and these include acetylcholine, certain lipids.
Genetics of Hormones:
Auxin Alters Gene Expression:
It has been known since long that auxin acts by inducing the expression of specific genes. Then it was suggested that the initial stimulation of growth by auxin was mediated by direct interaction with membrane components rather than by specific gene expression.
With the advent of molecular biology techniques it was possible to detect rapid changes in the levels of translatable mRNA i.e. mRNAs which are able to direct protein synthesis on ribosomes.
For some reasons, the level of translatable mRNA does not essentially correspond to the abundance of mRNA. Thus, specific cDNA probes have been employed in hybridization studies to measure abundance. So plus-and-minus screening methods have been used for the identification of auxin-regulated mRNAs.
It has been possible to clone cDNA sequences that are specifically stimulated by auxin. The rapid kinetics of auxin-induced gene expression suggests that gene expression may be involved in the rapid response to auxin.
It is an important attribute of signal transduction pathway which is initiated when the auxin binds to its receptor and then the activation of a select group of transcription factors. This factor enters the nucleus and promotes the expression of the specific genes. Genes whose expression is stimulated by the activation of pre-existing transcription factors are called primary response genes or early genes.
The expression of these genes cannot be blocked by any inhibitor of protein synthesis e.g. cycloheximide and implies that proteins required for auxin-induced expression of the early genes are present in the cell. Thus, time required for the expression of these genes is very short.
In animals the primary genes are shown to have three chief functions: encode proteins that regulate the transcription of secondary response genes or late genes; intercellular communication or cell-cell signalling, and another group of early genes is involved in stress adaptation.
Some workers have identified five major classes of early auxin-responsive genes i.e. Aux/IAA gene family, the SAUR gene family, the GH3 gene family, genes that encode glutathione S-transferase-like proteins, and genes that code 1-aminocyclo-propane-1-carboxylic acid (ACC) synthase.
Of these Aux/IAA family genes were the first to be discovered and encode short-lived transcription factors that function as repressors or activators of the expression of late auxin-inducible genes.
SAUR gene family is related to tropism, and are stimulated within 2-5 min of treatment and the response is insensitive to CH. SAUR expression is a convenient probe for the lateral transport of auxin during photo- and gravitropism.
GH3 early-family member genes are stimulated within 5 min of auxin application. Both Aux/IAA and GH3 are possibly the genes connected with required auxin-induced growth and other developmental processes. GST-genes appear to be stimulated by different environmental factors including heavy metals and various stress conditions.
It seems that their chief function is under stress conditions. Likewise, ACC synthase is also induced by stress, and possibly concerned with intercellular communication, especially during stress. Reporter genes have been used to identify auxin-response elements (AuxREs) within the promoters of the early auxin genes.
These AuxREs are usually combined in different ways to form auxin response domains within the promoter. Each auxin response domain is a composite structure composed of two AuxREs a variable constitutive element adjacent to a conserved TGTCTC element that confers auxin inducibility.
It may be stated that the expression of many of the early auxin genes are stimulated by CH and is accompanied by transcriptional activation and by mRNA stabilization.
It implies that the gene is being repressed by a short-lived repressor protein or by a regulatory pathway that involves a protein with a high turnover state. The data from CH suggest that a model for auxin action in which hormone blocks the interaction between the repressor and the activators seems prevailing.
Early auxin genes are insensitive to cycloheximide and indeed some genes are expressed and stimulated by CH and is accompanied by transcription activation and by mRNA stabilization.
Agravitropic Auxin Mutants Support the Cholodny-Went Model:
Agravitropic mutants do not respond to gravity. Mutagenized seedlings are allowed to grow along the surface of a vertically oriented agar plate, and plants whose roots do not grow straight downward are selected. In Arabidopsis several mutants are isolated.
Mutants initially isolated for their resistance to high auxin concentrations are shown to be agravitropic also, axrl and axr4, gravitropism is partially inhibited, while gravitropism already noted in axr2, axr3, auxl and,dwf is severely impaired.
AUX1 gene is already sequenced and shown to code for a protein similar to an amino acid permease. Taken together the data from these mutants, it is inferred that auxin is required for gravitropism-consistent with Cholodny-Went model.
Recently genetic evidence for the role of starch sheath in the perception of gravity in Arabidopsis was secured. A mutant, scarecrow (scr) in Arabidopsis had both the endodermis and starch sheath missing. Consequently the hypocotyl and inflorescence of the scr mutant are agravitropic though the root exhibits a normal gravitropic response.
On the basis of phenotypic appearance of scarecrow mutant it is concluded that: starch sheath is needed for gravitropism in shoot and, the root endodermis which lacks statoliths, is not required for gravitropism in roots.
In Arabidopsis, mutant which was starchless and two mutants with intermediate levels of starch, the wild type roots were more responsive to gravity than the intermediates, and the intermediates were more responsive than the starchless mutant.
Starchless mutants exhibit some gravitropism. Clearly, starch is required for a normal gravitropic response, starch independent gravity perception mechanism may also exist.
Starchless mutants in Arabidopsis may be explained on the basis of plasmalemma central control (PCC) model. Further data from Arabidopsis mutants revealed lack of lateral calcium gradient in horizontal root tips suggesting that calcium ions may not be required for gravitropism or the process may not be universal.
A series of Arabidopsis mutants name (alf)-aberrant lateral root formation have shown the role of auxin in the initiation of lateral roots. Thus the alfl mutant shows high proliferation of roots (adventitious and lateral) accompanied by 17-fold increase in endogenous auxin. alfl mutant had opposite phenotype and had no lateral roots and even lacked lateral root primordia and the lateral roots could not be induced through the application of auxin, alp is defective in the development of lateral roots primordia into mature lateral roots.
The arrested growth could be accomplished by the application of auxin exogenously. Thus in alf mutant study, it became evident that IAA is required ao at least two steps in the formation of lateral roots primordia: IAA is transported acropetally in the stele and is required to initiate cell division in the pericycle, and IAA is required to promote cell division and maintain cell viability in the developing lateral root.
In Arabidopsis plants an inhibitor which blocks polar auxin transport (NPA), causes the loss of gravitropism. If mature plants are treated with NPA, abnormal floral development is seen suggesting that polar auxin transport in the inflorescence is needed for normal floral development.
One mutant in Arabidopsis-pin-formed (pinl-l) was isolated whose flowers resembled NPA treated plants. This mutant showed different floral abnormalities e.g. wide petals, no stamens, lack of ovules, complete absence of floral bud etc. In such mutants polar transport of auxin was inhibited by 90% compared with wild type. Thus, main function of PINl gene may be to regulate polar auxin transport in the inflorescence.
Auxin may Induce Ubiquitination of Nuclear Proteins:
Arabidopsis mutants blocked at steps in the auxin response have provided useful information on the signal transduction pathway. A pathway has been discovered which is essential for auxin action and involves ubquitination of nuclear proteins. The first enzyme in this pathway is E1 which binds to and activates ubiquitin and uses ATP.
It has been demonstrated that in axrl (auxin-resistant), the gene involved results in many auxin responses (e.g., graitropism and gene expression) and was shown to encode an enzyme related to the N-terminal half of E1.
Some workers have succeeded in cloning and Arabidopsis homolog of the UBA2 gene called ECR1. The two proteins AXR1 and ECR1 form an E1-like heterodimer which binds to a family of small ubiquitin-related proteins called RUBs, and are related to ubiquitin.
This binding activates RUB and starts a pathway. This pathway is similar to the ubiquitination pathway in which RUB is covalently attached to a target protein. It may be mentioned that unlike ubiquitinated proteins, proteins tagged with RUB are usually activated rather than marked for destruction.
Studies in Arabidopsis have indicated that the AxR1-ECR1 heterdimer mediates the transfer of RUB to an enzyme E3 complex of the ubiquitination pathway, thereby activating the E3 complex.
It has been proposed that auxin enhances the activity of AXR1-ECR1. This is done either by inducing its synthesis or by activating a pre-existing enzyme. AXR1 protein is localized to the nuclei of dividing and elongating cells and thus it is suggested that auxin-induced ubiquitination occurs in the nucleus causing degradation of nuclear proteins by the 26S proteosome.
It is further assumed that repressor proteins that act as negative regulators of auxin-induced gene expression are potential targets of such a ubiquitination pathway.
Mutants and GA Biosynthesis Studies:
Mutants with defective GA biosynthesis have been isolated in several plants and are easy to locate being dwarf e.g. in rice, pea, Arabidopsis, etc.
These defective mutants are listed below:
(i) Maize d1 mutants responding to exogenous GA1but not GA20; these are defective in a relatively late step in GA biosynthesis, the 3B- hydrosylation of GA2o to form GA1.
(ii) Maize d5 mutant responding to ent-kaurene suggesting a much earlier block in GA biosynthesis. The defect is in the conversion of CPP to ent-kaurene catalyzed by ent-kaurene synthase B.
(iii) Ga4 mutant of Arabidopsis which shows SVT-KAURENE accumulation of 3P-hydroxylated GAs having been reduced indicating that GA4 may encode a 3β-hydroxylase.
(iv) In Arabidopsis, GA5, production of C19 GAs is reduced which indicates the role of GA5 in the oxidation of carbon-20 methyl group.
(v) In Arabidopsis GA1 locus having low levels of ent-kaurene synthase activity have been isolated and respond to exogenous ent-kaurene showing that its gene acts or regulates a much earlier step in GA biosynthesis.
Now several of these genes, have been cloned confirming the inferences drawn from physiological studies. There is a suggestion that ent-kaurene is synthesized in chloroplasts and that enzymes that convert it to GA12– aldehyde are microsomal but the precise mechanism of translocation of ent-kaurene from one organlle to another is still debated. Recently several genes for GA-biosynthesis enzymes have been isolated and characterized; copalyl diphosphate synthase (CPS, formerly ent-kaurene synthase A), ent-kaurene synthase (KS, formally ent-kaurene synthase B); GA 20-oxidase; GA 3β-hydrolase. Figure shown below is a simplified GA-biosynthesis pathway from tomato:
Recent studies have indicated the essentiality of GAs for the development of fertile flowers in tomato, and may also be required immediately after fertilization. In the GA-biosynthetic pathway the reactions catalyzed by GA 20-oxidases have been implicated as site of regulation. Three tomato GA 20-oxidases cDNA clones (Le20ox-1, -2, -3) were isolated. The three genes showed different organ specific patterns of mRNA accumulation.
Analyses of the transcript levels of the three GA 2-oxidases- genes as well as those of copalyl diphosphate synthase (LeCPS) and GA 3B-hydroxylase (Le 30H-2) during flower bud and early fruit development showed distinct patterns of mRNA accumulation. In the fully opened flower mRNA levels of Le20ox-1 transcripts was high but mRNA levels of Leox- 1, -2 and LeCPS were low.
These studies showed that transcrip levels of GA biosynthesis genes are highly regulated during flower bud development. The available data indicate that transcript levels of GA biosynthesis genes are highly regulated during flower bud development.
GA response mutants and signal transduction:
Single gene mutants damaged in their response to GA proved useful tools for the identification of genes that encode possible GA receptors or components of GA signal transduction pathways. Two classes of mutations were screened: GA-insensitive dwarfs and, constitutive GA responders.
The former class of mutants have been isolated from maize d8 and Arabidopsis gai mutants. Both these mutants resemble GA-deficient mutants barring that they do not respond to exogenous GA. They have been used for the study of receptor or signal transduction pathway.
Recently wild type CAl gene has been cloned, and it encodes a protein with nuclear localization signals. Further data have suggested that GAI gene acts as a repressor of GA responses and that GA can prevent this repression. The suggestion is that GA or its signal transduction component binds to the GAI protein and inactivates it.
The mutant gai gene may correspond to the site of binding to GA or signalling intermediate of GA. gai gene is shown to have a deletion of 17 amino acids and hence action of the repressor cannot be overcome by GA, and hence growth is inhibited. Moreover gai is semi-dominant mutation. Further data in pea mutants have indicated that the GA response could also be limited by deficiencies in one or more of the other hormones.
Slender mutants’ are comparable with wild type plants which have been Molecular biology and cytokinin-regulated processes:
ipt gene from Agrobacterium Ti plasmid has been introduced in several sp and this led to the synthesis of cytokinin. Introduction of ipt gene and its expression causes overproduction of this hormone in transgenics. These transgenics show several features that suggest the role played by cytokinin in plant physiology and development.
Some of these characters are: production of more leaves; leaves have high chlorophyll content and are greener; adventitous shoots formed from veins and petioles; leaf senescence retarded; apical dominance reduced; with increased cytokinin production plants become stunted; rooting of stem cuttings reduced and also root growth is reduced.
Some of the effects of cytokinin if controlled could enhance photosynthesis and productivity in crop species. In some sp transgenics with ipt were insect resistant.
Very few mutants which affect the synthesis or metabolism of cytokinin are reported. Some workers have identified mutants like ampl with changed meristem programe 1. This mutant was recessive and homozygous plants had altered phenotypic characters compared with wild type, but had more leaves, shoots, more infloresence branches, abnormal flowers.
The mutants had high levels of zeatin and dihydrozeatin. In fact cytokinin overproduction has been implicated in genetic tumors formation. Further experiments have demonstrated that auxin: cytokinin ratio was crucial for regulating morphogenesis.
Recent data have demonstrated direct involvement of cytokinins in cell cycle regulation. Further studies are needed to elucidate their precise mode of function in controlling cell cycle progression.
Cytokinins have also been shown to be natural regulator of leaf senescence. It will be interesting to find out how this hormone controls genes which express during leaf senescence or how their expression is regulated.
Two possible aspirants for a cytokines receptor have recently been identified and one fits into the steroid hormone receptor model-a cytosole receptor that migrates to the nucleus. The other candidate fits the membrane receptor model. Possibly both these proteins may be involved. Cytokinins are shown to regulate gene expression at a post-transcriptional step or may be concerned with increase in the abundance of specific mRNAs.
ABA exerts both short-term and long-term control over plant development. The latter effects are mediated by ABA-induced gene expression. ABA stimulates the synthesis of LEA/RAB/DHN family of proteins during seed development and during water stress.
These proteins possibly protect membranes and other proteins from desiccation damage. It has been possible to identify ABA-response elements and one of the transcription factors that binds to them. Evidence suggests the occurrence of both extracellular and intracellular ABA receptors in guard cells.
ABA closes stomata by causing long-term depolarization of the guard cell plasma membrane. Such depolarization is supposedly caused by an increase in Cacyt as well as alkalization of the cytosol.
Calcium increase is attributed to enhanced uptake and also its release from internal stores. The increase in calcium opens anion channels and result in membrane depolarization. The role of G-proteins in such a response is suggested. Possibly more than one signal transduction pathway may be involved in stomatal mechanism.