PLANT HORMONES SYNTHESIZED BY MICROORGANISMS AND THEIR ROLE IN BIOFERTILIZER-A REVIEW ARTICLE.

Shweta Sharma 1 and Mohinder Kaur 2 . 1. Department of plant pathology, Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan-173230. 2. Department of Basic Science (Microbiology), Dr Yashwant Singh Parmar University of Horticulture and Forestry, Nauni, Solan-173230. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Production and role of phytohormones:-There are two sources of phytohormones naturally available for the plants: endogenous production by the plant tissues and exogenous production by the associated microorganisms (Kumar and Lonsane, 1989; Arshad and Frankenberger, 1991;Costacurta and Vanderleyden 1995;Patten and Glick 1996). Many plant associated microorganisms are themselves capable of synthesizing phytohormones (given in table 1) which are necessary as mediator in communications between the plant host and its microflora. The ability to form plant hormones is believed to be a major property of rhizospheric, epiphytic and symbiotic bacteria that stimulate and facilitate plant growth called as plant growth promoting rhizobacteria (PGPR) strains (Kameneva and Muronets, 1999;Karvchenko et al., 2004;Suzuki and Oyaizu, 2003). On the other hand, certain free-living microorganisms (i.e. those that form no association with plants in the course of the life cycle) are also capable of synthesizing plant hormones.  (Furukawa,1996; Thakur, and Vyas, 1983) 2. The conversion of tryptophan into indole-3-acetic acid aldehyde may involve an alternative pathway in which tryptamine is formed. This pathway is believed to operate in pseudomonades and azospirilla, anundentified mycorrhizal fungus of orchid (Ophrys lutea Cav.) (Barroso, 1986) and the cyanobacterium Chlorogloea fritschii (Ahmad, and Winter, 1969). 3. IAA biosynthesis via indole-3-acetamide formation takes place in phytopathogenic bacteria Agrobacterium tumefaciens, Pseudomonas syringe and E. herbicola; certain streptomycetes; saprophytic pseudomonades Pseudomonas putida and Pseudomonas fluorescence. 4. IAA biosynthesis that involves tryptophan conversion into indole-3-acetonitrile is found in plant, Alcaligenes fecalis and possibly, the cynobacterium Synechocystis sp. 5. The tryptophan-independent pathway, more common in plants, is also found in microorganisms (Azospirillium and Cynobacteria) However, the contribution of this pathway to IAA biosynthesis is insignificant and the mechanisms are largely unknown. In plant, IAA binds to sugars, amino acids and protein, forming storage (inactive) forms which release the phytohormone when it is needed (the physiological activity is recovered shortly thereafter). Omission of tryptophan from the culture medium decreases the level of IAA synthesis by the culture's microorganisms. Exogenous tryptophan may augment auxin biosynthesis by an order of magnitude or higher, this being the reason why the yield of the phytohormone in the most active strains exceeds 80-100mg IAA per 1 ml culture medium (Tsavkelova et al.,2003).

Gibberellins:-
A substance including excessive extension of rice sprouts, first isolated from the phytopathogenic fungus Fusarium moniliforme in the 1930s, was given the name gibberellins (after the perfect stages of Fusarium monilifomra, Gibberella fujikoroi) (Escamilla et al.,1999). Gibberellins, classified with diterpenes, consist of isoprene residues that usually form four rings (A, B, C and D). Gibberellic acids (GAs), GA 3 , GA 7 , GA 1 , and GA 4 are the best studied phytohormones of this group; they exhibit maximum biological activity and are the most widespread in nature. Gibberellins amount to more than 100 compounds, constituting the largest class of phytohormones, which are found 1757 in both plants and microorganisms. Certain compounds are classified with gibberellins based solely on their characteristic biological activity (they have a different structure). First and foremost, gibberellins affect the division and elongation of the cells constituting the intercalary meristem, although stimulation of fluorescence, activation of the synthesis of membranes and amylolytic enzymes, and other effects have also been described. The Gibberellins (GAs) are complex molecules of tetracarbocyclic diterpernes. GAs numbering is not related to their structure. Molecules, whose structure has been elucidated, are numbered in approximate order of their discovery. There is continuing interest in the biosynthic origin of the GAs since some of them have important activities in plants. The most important GA in plant is GA 1 , primarily responsible for stem elongation. In Gibberellia, GAs biosynthesis is catalyzed by enzymes falling into three classes: terpene cyclases catalyze the synthesis of ent-kaurene from geranylgernayl diophosphate; cytochrome P450 monooxygenase catalyze the steps of the pathway from ent-kaurene to GA 12 ; and soluble dioxynenase catalyze the final steps of the pathway. The ability of Azospirillum lipoferum and Azospirillum brasilense to metabolize GA 20 GA 1 in rice (Oryza sativa L.) seedlings suggests that an enzyme similar to that operating in plants (2-oxyglutarate-dependent dioxygenase) is involved in gibberellins biosynthesis in these bacteria (belonging to the genera Azotobacter, Pseudomonas and Lactobacillus) certain yeast strains, and mycelia rhizospheric fungi.GA increases the growth rate and promotes nitrogen fixation in cyanobacteria of the genus Anabaena and it also stimulates the formation of lytic enzymes in certain bacteria and fungi (Vinklarkova and Sladky,1978;Barea,1974 ).

Cytokinins:-
Cytokinins are adenine derivatives. The first compound exhibiting cytokinin activity was isolated from the semen of herring. Subsequently, a factor responsible for control of cell division was isolated from maize (Zea mays L.); hence named as zeatin. The second natural cytokinin to be identical was isopentenyadenine; this compound was a minor base in serine tRNA of yeast. Studies with slime mold Dictyostelium discoideum revealed that 5'-AMP was a direct precursor of isopentenyl adenosine 5'-phosphate. The enzyme catalyzing this conversion, dimethylallyl diphosphate: 5'-AMP transferase (or isopentenyl transferase) was also found in cell-free extract from maize kernels, and from tobacco callus tissue cultures that became cytokinin-autonomous. Recently several genes encoding the isopentenyl transferase have been identified from Arabidopsis thaliana. A corresponding enzyme from the bacterium Agrobacterium tumefaciens, encoded by the ipt gene, has been studied in depth at the molecular level, and the same gene was also found in Pseudomonas syringae pv. savastanoi, where it is named ptz.  The synthesis of cytokinins is tRNA -dependent in Pseudomonas aeruginosa, Rhizobium spp., Rhodococcus fascians, and the fungus Taphrina cerasi (Gray, 1996). In the majority of cases, however, tRNA degradation 1758 produces inactive cis-isomers of zeatin, whereas the active trans-isomers are formed by de novo biosynthesis. On the other hand, tRNA of the diazotropic bacterium Azotobacter vinelandii was found to contain 2-methylthioribosyltrans-zeatin.
Ethylene:-Ethylene biosynthesis by plants originates from methionine. The first step is the synthesis of S-adenosyl-methionine, followed by its conversion into 1-aminocyclopropane-1-carboxylic acid (ACC). ACC is the direct precursor of ethylene. The ACC oxidase, formerly known as the ethylene-forming enzyme (EFE), was first characterized in apple. Ethylene production has been also reported for bacteria and fungi.
Phytohormone-like substances formed by microorganisms affect not only the plant host, but also the producer microorganisms, which undergoes the necessity of holistic approaches to the plant and its associated micro biota as components of a single system. Some researchers believe that excretion of IAA by bacteria grown under unfavorable condition may have considerable functional importance as a factor increasing the probability of forming plantmicroorganism association. This conclusion based on the finding that the amount of IAA reaches maximum values during the steady-state stage of development, characterized by nutrient depletion. The role of hormones as regulatory substances should therefore be viewed on abrader scale, because they act as intermediaries not only in processes confined to plant tissues, but also in communications of diverse organisms inhabiting the same ecological niche. Each participant of such a community has an intrinsic biochemical activity and pursues its own ends; both pathogenic and symbiotic microorganisms, however, excrete the same phytohormones. The difference in the resulting effect is not infrequently reduced to the concentration of a phytohormone. In this particular this case, microorganisms populating the root surface and capable of excreting phytohormones gets advantages in its colonization (Maor et al., 2004).

Thus inoculation with
Azospirillum mimics typical growth response induced by auxins, which are inhibitory of plant growth at high concentrations and stimulatory at lower levels. 3. Use of plant hormone producer microorganisms as inoculum for many crops:-Microbial phytohormones exert beneficial effects when plant seeds, seedlings etc. are treated with cultures and /or suspensions of producer microorganisms. Seeds treatment with soil rhizobacteria Azospirillum, Bejerinckia, Rhizobium, Agrobacterium, Bacillus, Pseudomonas, Mycobacterim, Arthrobacter, Methylovorus, and Flavobacterium strongly stimulates the germination capacity and germination in seed, also increasing the growth and crop productivity in mature plants (Dileep,1998). The augmentation of the growth rate correlates with the increase in ability of the bacteria to colonize the plant and the amount of the phytohormones formed (Zvyagintsev, 1995). Strains of rhizobacteria producing small amounts of auxins increased considerably the development of wheat (Triticum aestivum L.) and its crop productivity. Inoculation with cytokinin-producing methylobacteria of transgenic tobacco plants characterized by altered morphology (rootlessness) restored root formation and the effects of the microorganism culture on seed germination and plant development were similar to those of the phytohormones or the culture liquid of methylobacteria. Treatment of dwarf rice (which lacks the ability to synthesize gibberellins) with Azospirillum lipoferum and A. brasilense resulted in a pronounced stimulation of plant growth. This effect was due to the ability of the bacteria to metabolize exogenous GA20 (gibberellins 20) into the biologically active GA1 (Tudzynski, 1999). Moreover, , industrial production of gibberellins for agriculture relies primarily on the cultivation (on an industrial scale) of the fungus Fusarium moniliforme ,the perfect stage of which (Gibberella 1759 fujikoroi) produces considerable amounts of diverse gibberellins. (Polyanskaya et al., 2002). Bacterial treatment of seed makes it possible to achieve germination of germination-resistant seed of rare, decorative or industrially important plants. Thus, bacteria of the genera Pseudomonas, Bacillus, Xanthomonas, Rhodococcus and Micrococcus, which all form auxins, strongly stimulate symbiotic germination of the seeds of tropical orchids and accelerate their development under greenhouse conditions. It should be taken into account that the beneficial effects of bacterial treatment depend on a variety of factors, including the activity of the strain, the concentration of the cells, the amount of phytohormones in the culture liquid , the quantity of the dry preparation of the stimulating microorganism, the duration of the treatment, the species of the plant ,the state of the indigenous microflora at the time of seeding , the characteristics of the soil and the general conditions of the agro technological complex. The introduction of bacterial inoculums is more successful if the strains are isolated from the rhizoplane or rhizosphere of mature plants of the same species (Lalande et al., 1989). It is not frequent that stimulation of plant growth and development by PGPR (plant growth promoting rhizobacteria) strains of bacteria is underlain not only by phytohormone formation, but also by their capacity for nitrogen fixation, improvement of plant nutrition (water and mineral) and prevention or suppression of phytopathogen growth; the latter effect is due to excretion by PGPR strains of bactericidal and fungicidal substances (Glick and Pasternak, 1998). 4. Sugar cane promotion:-Up to 80% of the total N incorporated into several sugar cane cultivars can be attributed to BNF (biological nitrogen fixation). In addition, the growth promotion can be driven by a hormonedependent mechanism. Under N-sufficient growth condition, plants inoculated with Gluconacetobacter diazotrophicus, either as the wild type or a nifD mutant are approximately 20% taller than non inoculated plants. These results suggested that Gluconacetobacter diazotrophicus could benefit sugarcane by two ways: by transfer of bacterial nitrogen fixed and as well as via phytohormones production (Sevilla et al, 2001). 5. Gain in root length associated to ACC-deaminase:-Ethylene plays an inhibitory role on root elongation. A role for the ACC-deaminase in preventing ethylene effect was shown in inoculation experiments of canola roots by E.cloacae. The plant growth promotion effect is linked to the lowering of plant ethylene levels by the bacterial ACC-deaminase.

Conclusion:-
Understanding of IAA, Gibberellins and cytokinins metabolism calls for further identification and analysis of the intermediates, enzyme and genes involved in their biosynthesis, as well as in the isolation of mutants defective in each pathway. Although the production of phytohormones at the free living state is well established in many microorganisms, there is still insufficient evidence for their synthesis in their natural habitats. The ecological significance of phytohormones production by bacteria would be more convincing if it could be demonstrated that bacterial phytohormones production occurs while bacteria colonize the root system. As both the plant and the bacteria synthesize and secrete auxins, gibberellins and cytokinins is difficult to address the contribution of one particular hormone as responsible of the effects observed. Thus the possibility that the host plant directs the bacterium to produce IAA through Trp present in root exudates is intriguing and speculative at this point.
Plant-associated microflora is the richest source of microorganisms synthesizing phytohormones. The bulk of evidence shows that phytohormones formed by fungi, algae and bacteria are structurally identical to their plant counterparts. In future, the use of transcriptional (or other type) fusion for the analysis of the differential expression of the bacterial genes involved in phytohormones biosynthetic pathways in association with the host plant should generate important information.
In recent years, a number of studies on inoculation of cereal crops (wheat, maize, sugar cane, sorghum and sunflower) and horticulture crops with PGPR have been performed. Beneficial effects such as increases in nitrogen content and yield have been reported in Belgium, Israel, France, Argentina, Uruguay, Mexico, USA and South Africa. Success of field experiments depends of many parameters, such as the strain used, concentration of bacterial inoculum, viability of bacteria during storage, carrier employed, appropriate inoculation methodology, and soil characteristics. The identification of many traits and genes related to the beneficial effects of inoculated bacteria shall result in a better understanding of the performance of bioinoculants in the field. It will also provide a strategy to design genetically modified strains with improved PGP effects. This multiplicity of effects of phytohormones determines the function of the plant-microorganism community as a whole. The productive efficiency of a specific PGPR may be further enhanced with the optimization and acclimatization according to the prevailing soil conditions. In future, they are expected to replace the chemical fertilizers, pesticides and artificial growth regulators which have numerous side-effects to sustainable agriculture. Further research and understanding of mechanisms of 1760 PGPR mediated-phytostimulation would pave the way to find out more competent rhizobacterial strains which may work under diverse agro-ecological conditions.