Ördög Vince, Molnár Zoltán (2011)
Debreceni Egyetem, Nyugat-Magyarországi Egyetem, Pannon Egyetem
Hormones and other regulatory chemicals are now used in a variety of applications where it is desirable for commercial reasons to control some aspect of plant development.
Commercial application of auxins
Auxins have been used commercially in agriculture and horticulture for more than 50 years. The synthetic auxins are used in commercial applications largely because they are resistant to oxidation by enzymes that degrade IAA. In addition to their greater stability, the synthetic auxins are often more effective than IAA in specific applications. One of the most widespread uses of auxin encountered by the consumer is the use of 2,4-D in weed control. 2,4-D and other synthetic compounds, such as 2,4,5-T and dicamba, express auxin activity at low concentrations, but at higher concentrations are effective herbicides.
Indolebutyric acid and naphthaleneacetic acid are both widely used in vegetative propagation – the propagation of plants from stem and leaf cuttings. This application can be traced to the propensity for auxin to stimulate adventitious root formation. Generally marketed as “rooting hormone” preparations, the auxins, usually a synthetic auxin such as NAA or IBA, are mixed with an inert ingredient such as talcum powder. Stem cuttings are dipped in the powder prior to planting in a moist sand bed in order to encourage root formation.
4-CPA may be sprayed on tomatoes to increase flowering and fruit set while NAA is commonly used to induce flowering in pineapples. This latter effect is actually due to auxin-induced ethylene production. NAA is also used both to thin fruit set and prevent preharvest fruit drop in apples and pears. These seemingly opposite effects are dependent on timing the auxin application with the appropriate stage of flower and fruit development (Figure 3.25). Spraying in early fruit set, shortly after the flowers bloom, enhances abscission of the young fruits (again, due to auxin-induced ethylene production). Thinning is necessary in order to reduce the number of fruits and prevent too many small fruits from developing. Spraying as the fruit matures has the opposite effect, preventing premature fruit drop and keeping the fruit on the tree until it is fully mature and ready for harvest.
The use of synthetic auxins, especially the chlorinated forms, as herbicides has come under close scrutiny by environmental groups because of potential health hazards. 2,4,5-T, for example, has been banned in many jurisdictions because commercial preparations contain significant levels of dioxin, a highly carcinogenic chemical.
Figure 3.25 Auxin promotes fruit development that produced by achenes (source: Taiz L., Zeiger E., 2010)
Commercial use of gibberellins
The major uses of gibberellins (GA3), applied as a spray or dip, are to manage fruit crops, to malt barley, and to increase sugar yield in sugarcane. In some crops a reduction in height is desirable, and this can be accomplished by the use of gibberellin synthesis inhibitors.
Many of the table grapes grown in the United States are a genetically seedless variety that would naturally produce small fruit on very compact clusters. Almost all seedless grapes on the market are treated with GA3. It substitutes for the presence of seeds, which would normally be the source of native GAs for fruit growth. Repeated spraying with GA3 increases both rachis length (producing looser clusters) and fruit size (Figure 3.26). The increased rachis length prevents the cluster from being too compact, and this reduces the chance of fungal growth inside the cluster. Two to three additional applications of GA3 during fruit development are thought to increase berry size by enhancing the import of carbohydrates into the developing fruit.
Figure 3.26 Gibberellin induces growth in Thompson’s seedless grapes (left – control, right – sprayed with GA3) (source: Taiz L., Zeiger E., 2010)
Gibberellic acid is also used to boost cherry production. Sweet, bing cherries are sprayed 4 to 6 weeks before harvest to increase fruit size. Application of GA3 to tart cherries increases yield through enhanced bearing. Gibberellin A4 (GA4) is used to promote the fruit set of apple and pear trees. For example, in some apple cultivars the amount of fruit produced is often limited by biennial bearing, a phenomenon whereby the production of a heavy crop of fruit one year inhibits the subsequent production of flower buds, and hence, the yield of fruit the following year. The alternate bearing of some cultivars can be overcome by applying GA4 in the “off” year to promote the formation of flower buds, and subsequent fruit set. In regions of Europe where fruit set of apple and pear trees is often reduced by inclement weather at the time of pollination, the application of a hormone mixture can promote the production and subsequent growth of parthenocarpic (seedless) fruit. GA4/7 is also used on Golden Delicious apples to prevent abnormal cell divisions in the epidermal layer that produce “russetting”. Gibberellic acid is also applied to citrus crops, though the actual use depends on the particular crop. For example GA3 is sprayed onto oranges and tangerines to delay or prevent rind-aging, so that fruit can be harvested later without adverse effects on rind quality and appearance. For lemons and limes, GA3 synchronizes ripening and enhances fruit size.
Gibberellins from the embryo of germinating grains are necessary for the synthesis of α-amylase by the cells of the aleurone layer, which, in turn is necessary for the hydrolysis of starch within the endosperm. In the brewing industry, the production of beer relies on this hydrolytic breakdown of starch in barley grains to yield fermentable sugars, principally maltose, which are then subjected to fermentation by yeast. During fermentation, glycolytic enzymes from yeast break down the sugars, resulting in ethanol. In the multistep malting process, mature barley grains are steeped or soaked to allow them to imbibe water. Next, the grains are spread out to germinate, during which time the starch within the endosperm will be hydrolyzed by α-amylase allowing the embryo to begin to grow. This process of starch breakdown is referred to as “modification”. Gibberellic acid may be applied during this time and will supplement the native GAs in the grain, enhance the production of α-amylase, and consequently, speed up the hydrolysis of starch.
Manipulation of cytokinins is a tool to alter agriculturally important strains
Some of the consequences of altering cytokinin function could be highly beneficial for agriculture if synthesis of the hormone can be controlled. Because leaf senescence is delayed in the cytokinin-overproducing plants, it should be possible to extend their photosynthetic productivity. Indeed, when an ipt gene is expressed in lettuce from a senescence-inducible promoter, leaf senescence is strongly retarded, similar to the results observed in tobacco.
In addition, cytokinin production could be linked to damage caused by predators. For example, tobacco plants transformed with an ipt gene under the control of the promoter from a wound-inducible protease inhibitor II gene were more resistant to insect damage. The tobacco hornworm consumed up to 70% fewer tobacco leaves in plants that expressed the ipt gene driven by the protease inhibitor promoter.
Manipulation of cytokinin also has the potential to increase grain yield in rice. Humans have unwittingly taken advantage of the promotive effect of cytokinin on the shoot apical meristem in their breeding of cultivated rice varieties. The rice varieties japonica and indica differ dramatically in their yield, with the latter generally producing more grains in their main panicle and ultimately a higher yield. The increased grain number in indica varieties has recently been linked to a decrease in the function of a cytokinin oxidase gene. As a consequence of the reduced function of this cytokinin oxidase in the indica varieties, cytokinin levels are higher in the inflorescence, which alters the inflorescence meristem such that it produces more reproductive organs, more seeds per plant, and ultimately a higher yield (Figure 3.27).
Figure 3.27 Cytokinin regulates grain yield in rice (indica variety has low number of cytokinin oxydase genes) (source: Taiz L., Zeiger E., 2010)
Large-scale cloning of plants by micropropagation
With a relatively small investment in space, technical support, and materials, tissue culture has made it possible to produce literally millions of high-quality, genetically uniform plants. The process is known as micropropagation. The most common technique is to place excised meristematic tissue on an artificial medium containing a cytokinin/auxin ratio that reduces apical dominance and encourages axillary bud development. The new shoots can be separated and sub-cultured to produce more axillary shoots, or placed on a medium that encourages rooting. Once roots appear, the plantlets can be planted out and allowed to develop into mature plants. Alternatively, excised tissues can be used to establish callus cultures, which may then be induced to form roots and shoots by manipulating the cytokinin/auxin ratio.
Micropropagation can also be an effective way to eliminate viruses and other pathogens and produce commercial quantities of pathogen-free propagules. The first plants to be mass-produced by tissue culture were virus-free orchids of the genus Cymbidium, but the technique has also been found useful for potato, lilies, tulips, and other species that are normally propagated vegetatively. Potato, for example, is vegetatively propagated through buds on the tubers, a system that readily transmits viruses to the next generation. Micropropagation of potato from meristem cultures has proven to be an effective way to isolate virus-free lines.
Micropropagation is also used extensively in the production of forest tree species. Here the propagules are generated primarily from cultures of axillary and adventitious buds; callusing and differentiation of new buds is rarely used. A similar approach has been applied successfully to cultivars of apple (Malus), peach (Pyrus), and pear (Prunus). Because most temperate fruits are highly heterozygous, they do not breed true from seed but are propagated by vegetative cuttings. Rooting of microcuttings in culture is now a routine procedure in many commercial laboratories.
The use of ethylene and brassinosteroids in plant production
As ethylene regulates many physiological processes in plant development, it is one of the most widely used plant hormones in agriculture. Auxins and ACC can trigger the natural biosynthesis of ethylene and in several cases are used in agricultural practice.
Ethephon (Ethrel) is the most widely used ethylene releasing compound
Because of its high diffusion rate, ethylene is very difficult to apply in the field as a gas, but this limitation can be overcome if an ethylene-releasing compound is used. The most widely used such compound is Ethephon, or 2-chloroethylphosphonic acid, which was discovered in the 1960s and is known by various trade names, such as Ethrel. Ethephon is sprayed in aqueous solution and is readily absorbed and transported within the plant. It releases ethylene slowly by a chemical reaction, allowing the hormone to exert its effects. It is used for:
hastening fruit ripening of apple, tomato, and degreening of citrus;
synchronized flowering and fruit set in pineapple, and accelerated abscission of flowers and fruits;
inducing fruit thinning or fruit drop in cotton, cherry, and walnut;
promoting female sex expression in cucumber, to prevent self-pollination and increase yield;
inhibition of terminal growth of some plants in order to promote lateral growth and compact flowering stems.
Brassinosteroid (BR) application to crop plants is most effective under stress conditions
Brassinosteroids were discovered as a class of growth promoting hormones, and researchers immediately recognized their potential applications to agriculture. For the past 20 years, numerous small-scale studies have been conducted to test the ability of BRs to increase yields of crop plants. BL has been found to increase bean crop yield (based on the weight of seeds per plant) by about 45%, and to enhance the leaf weight of various lettuce varieties by 25%. Similar increases in the yields of rice, barley, wheat, and lentils have been observed. BL also promoted potato tuber growth and increased its resistance to infections. Tomato fruit set was also enhanced by BL. In addition to such small-scale studies, large-scale field trials using brassinosteroid derivatives have now been conducted in Japan, China, Korea, and Russia. The results of the field trials have been highly variable and appear to reflect the degree of stress under which the crop was grown. A crop grown under optimal conditions shows little effect of applied BR, while a crop grown under conditions of stress shows dramatic effects of BR application on yield.
Microbial plant hormones
Bacterial and fungal plant hormones
Some bacteria and fungi are intimately associated with higher plants. Many of these microorganisms produce and secrete substantial amounts of cytokinins and/or cause the plant cells to synthesize plant hormones, including cytokinins. The cytokinins produced by microorganisms include trans-zeatin, iP, cis-zeatin, and their ribosides, as well as 2-methylthio-derivatives of zeatin. Infection of plant tissues with these microorganisms can induce the tissues to divide and, in some cases, to form special structures, such as mycorrhizal arbuscules, in which the microorganism can reside in a mutualistic relationship with the plant.
In addition to the crown gall bacterium, Agrobacterium tumefaciens, other pathogenic bacteria may stimulate plant cells to divide. Without Agrobacterium infection, the wound-induced cell division would subside after a few days and some of the new cells would differentiate as a protective layer of cork cells or vascular tissue. However, Agrobacterium changes the character of the cells that divide in response to the wound, making them tumorlike. They do not stop dividing; rather, they continue to divide throughout the life of the plant to produce an unorganized mass of tumorlike tissue called a gall (Figure 3.28).
Figure 3.28 Tumor that formed on a tomato stem infected with the crown gall bacterium bearing cytokinin biosynthesis genes (source: Taiz L., Zeiger E., 2010)
Increased cytokinin, supplied by interacting bacteria, fungi, viruses, or insects, can cause an increase in the proliferation of the shoot apical meristem and/or the growth of lateral buds, which normally remain dormant. This proliferation, known as fasciation. often manifests as a phenomenon known as a witches' broom, so-called because these growths can resemble an old-fashioned straw broom. One well-studied causative agent of fasciation is Rhodococcus fascians. R. fascians produces several different cytokinins, including both cis-and trans-zeatin as well as their 2-methylthio-derivatives. This mixture of cytokinin species acts synergistically through the host's normal cytokinin signaling pathway to alter host development. R. fascians also secretes the auxin IAA, which contributes to the alteration in the growth of the host plant. Fasciation, which can also arise spontaneously by a mutation, is the basis for many of the horticultural dwarf conifers.
Microalgal plant hormones
There is accumulating evidence that both cyanobacteria and microalgae like to many seaweeds produce plant hormones, or demonstrate plant hormon-like activity. Recently, it is quite often that the beneficial effects of nitrogene-fixing cyanobacteria are explained with the influence of their PGRs instead of the increased available nitrogen for the rice plants.
The possibilities for applying microalgae in crop production has been investigated at the Faculty of Agricultural and Food Sciences, University of West Hungary, in Mosonmagyaróvár for several years. Indicator plants like potatoes and sugar beet proved the applicability of 3 algal strains out of many others, which we isolated (MACC-6, MACC-116, MACC-612). Small plot trials were carried out at ecological districts of the country, which show considerable differences, e.g. in counties Komárom, Szabolcs and Csongrád. We managed to influence the process of crop yielding capacity of potato and sugar beet with the investigated algal strains differently in method and size per habitat and year. We were able to influence the time of tillering and tuber building, the number and size of tubers, which resulted in yield increase. At one of the trial sites in county Csongrád the strain MACC-612 showed a definite and well recognisable fungicide side effect in potatoes. We were able to influence the competition between beetroot and the foliage of sugar beet significantly and as a result of a longer active foliage life we could avoid harmful change of leaves even in climatic stress situations. With this successful treatment sugar beet yield per area unit increased and although the sugar content in percentage slightly decreased the absolute sugar yield increased as well (Figure 3.29). We applied microalgae in potato trials alone but they were applied as combination partners of fungicides in sugar beet. As a result the strains MACC-116 and MACC-612 can especially well be combined with strobilurin preparations.
Figure 3.29 Sugar beet treatments with microalgae increase the sugar yield (source: own result)
Compounds of natural origins derived from higher plants are widely used in disease and pest control in ecological production. It is known, that seaweeds also contain chemical constituents, which has antimicrobial properties. In our experiments 255 microalgae strains were examined in vitro in agar gel diffusion test, to establish their effect on growth and development on plant pathogenic fungi. Four percent of tested algae strains showed fungicide, 59% fungistatic activity at least against one plant pathogen. The most effective strains were examined against a biotrophic plant pathogen, Plasmopara viticola a causal agent of grapevine downy mildew in vitro, using leaves and leaf discs. Inhibition effect of algae extract on the sporulation of pathogen reached the 100%. A field experiment was conducted in 2002, where MACC-14 strain was applied in 3, 5 and 10mg/ml concentration. The efficacy of algal suspension was about 50%.
In the tissue cultures of pea and tobacco the combination of extracellular compounds from microalgae and synthetic PGRs produced more fresh weight and regenerated shoot numbers than the control. The dilution of freeze dried biomass derived from MACC-304 and 612 has the same beneficial effect as the synthetic PGRs on tissue cultures of peas and tobacco.
According to the above mentioned own results we can state:
bacteria, microalgae and cyanobacteria are able to produce several types of plant hormones;
physiological status of cells (cell cycle) and environmental factors (light) influence the hormone production;
highly reproducible results can be achieved by using synchronous cultures of microalgae, which can also explain the function of plant hormones in microalgae;
broad leaf plants respond with yield increase on microalgal treatments.
Other synthetic growth regulators
Antiauxins inhibit the effects of auxins found in plants
Antiauxins are another class of synthetic auxin analogs. These compunds, such as α-(p-chlorophenoxy) isobutyric acid or PCIB, have little or no auxin activity but specially inhibit the effects of auxin. When applied to plants, antiauxins may compete with IAA for specific receptors, thus inhibiting normal auxin action. One can overcome the inhibition of an antiauxin by adding excess IAA.
Several compounds have been synthesized that can act as auxin transport inhibitors, including NPA (l-N-naphthylphthalamic acid), TIBA (2,3,5-triiodobenzoic acid), CPD (2-carboxyphenyl-3-phenylpropane-l,3-dione), NOA (l-napthoxyacetic acid), 2-[4-(diethylamino) -Z-hydroxybenzoyl] benzoic acid, and gravacin. NPA, TIBA, CPD, and gravacin are auxin efflux inhibitors (AEIs), while NOA is an auxin influx inhibitor. Some AEIs, such as TIBA, have weak auxin activity and inhibit polar transport in part by competing with auxin at the efflux carrier site. Other AEIs, such as CPD, NPA, and gravacin interfere with auxin transport by binding to a regulatory site. Some inhibitors, such as gravacin, interfere more specifically with one type of transporter, while others, such as NPA, bind to and interfere with multiple proteins, some of which are only indirectly involved in auxin transport. Some natural compounds, primarily flavonoids, also function as auxin efflux inhibitors.
Synthetic antiauxins are used for:
inhibition of shoot development of stored onions and potato tubers;
inhibition of axillary shoot development in tobacco;
control (inhibition) of lawn growth;
promotion of sugarcane ripening;
prevention against Fusarium diseases;
promotion of stooling in cereals.
The inhibition of gibberellin biosynthesis also has commercial applications
The inhibition of gibberellin biosynthesis also has commercial applications. The growth of many stems can be reduced or inhibited by synthetic growth retardants or antigibberellins. These include AMO-1618, cycocel (or, CCC), Phosphon-D, ancymidol (known commercially as A-REST), and alar (or, B-nine). Growth retardants mimic the dwarfing genes by blocking specific steps in gibberellin biosynthesis, thus reducing endogenous gibberellin levels and suppressing internode elongation. These compounds have found significant commercial use, particularly in the production of ornamental plants. Growth retardants may be applied to potted plants either as a foliar spray or soil drench. Their principal effect is to reduce stem elongation, resulting in plants that are shorter and more compact, with darker green foliage. Flower size, however, is unaffected. Commercial flower growers have found these inhibitors useful in producing shorter, more compact poinsettias, lilies, and chrysanthemums, and other horticultural species. In some areas of the world, wheat tends to “lodge” near harvest time, that is, the plants become top-heavy with grain and fall over. Spraying the plants with antigibberellins produces a shorter, stiffer stem and thus prevents lodging. Antigiberellins also have been used to reduce the need for pruning of vegetation under power lines.
Inhibition of ethylene production and promotion preservation of fruits
Storage facilities developed to inhibit ethylene production and promote preservation of fruits have a controlled atmosphere of low O2 concentration and low temperature for the inhibition of ethylene biosynthesis. A relatively high concentration of CO2 (3 to 5%) prevents ethylene's action as a ripening promoter. Low pressure (vacuum) is used to remove ethylene and oxygen from the storage chambers, reducing the rate of ripening and preventing overripening. The ethylene binding inhibitor Ethylbloc® is increasingly being used to extend the shelf life of various climacteric fruits. Specific inhibitors of ethylene biosynthesis and action have proven useful in the postharvest preservation of flowers. Silver (Ag+) has been used extensively to increase the longevity of cut carnations and several other flowers. The potent inhibitor AVG retards fruit ripening and flower fading, but its commercial use has not yet been approved by regulatory agencies.
Decreased brassinosteroid (BR) synthesis or signaling lead to increased biomass and final seed yield
Reduced BR function can contribute to agriculture as well. For example, decreased BR synthesis or signaling in rice results in dwarfed plants with an erect leaf habit, which allows higher planting densities, leading to increased biomass and final seed yields. As researchers continue to explore BR's effects on plant development, additional applications of brassinosteroids to agriculture are bound to emerge.