Phytohormone Synthesis and Perception

Flower colour and scent of ornamental crops are important candidate target traits for genetic manipulation with biotechnology tools because they are the obvious reasons that these crops are grown. However, there has been much research attention given to the manipulation of phytohormone synthesis and perception in ornamental crops due to the wide range of physiological processes in which they are involved. Currently, several issues in crop production and post-harvest handling in the ornamental horticulture industry reflect a need to be able to understand physiological processes that are normally controlled by plant hormones. For example, significant effort and money is spent applying chemicals to plants to control the synthesis of gibberellic acid (GA) and subsequent plant height during crop production. In many potted flowering crops and most bedding plant crops, as many as three to five applications of growth-regulating compounds may be used during a crop cycle. This adds a significant amount of production cost for chemicals and labour to apply and handle them, while being a negative factor environmentally. In addition, many of the newly introduced ornamental species that have not been in commercial production for a long period of time are receiving particular attention in conventional breeding and selection for dwarf plants because their natural habit does not fit into the mass-market operation scheme requiring compact plants that fit into transport vessels. It is apparent that the manipulation of endogenous GA concentration in plants through genetic engineering has the potential to produce ornamental and flowering plants with a diverse array of reduced-height phenotypes.

Considerable research over the past two decades has been conducted to isolate GA biosynthesis mutants (for review, see Hedden and Proebsting, 1999) and to elucidate the GA biosynthetic pathway (for review, see Hedden and Kamiya, 1997; Hedden and Phillips, 2000). The initial cloning and characterisation of gibberellin 20-oxidase genes from Arabidopsis (Phillips et al., 1995) and spinach (Wu et al., 1996) provided some of the first practical molecular tools needed to develop plants with altered endogenous GA levels. In Arabidopsis thaliana, GA 20-oxidase catalyses consecutive steps late in GA biosynthesis, and is encoded by three genes with differential patterns of expression (Phillips et al., 1995; Coles et al., 1999). Over-expression of GA 20-oxidase in Arabidopsis leads to increased production of active GA (GA4) and subsequent stem elongation, while expression of antisense GA 20-oxidase leads to reduced levels of active GAs and reduced rates of stem elongation (Coles et al., 1999). Heterologous over-expression of a pumpkin GA 20-oxidase gene driven by constitutive promoters in both transgenic Solanum dulcamara (Curtis et al., 2000) and lettuce (Lactuca sativa ‘Vanguard’) (Niki et al., 2001) also resulted in the production of dwarf plants. In both cases, endogenous concentrations of active forms of GA, such as GA1 and GA4 were reduced in transgenic dwarf plants, while concentrations of various inactive forms of GA such as GA25 were increased, indicating a diversion of the normal pathway of GA biosynthesis towards the production of inactive forms of GA.

More recently, additional tools for use in engineering dwarf plants have been developed as a result of the cloning and characterisation of genes involved in the regulation of GA degradation. The first steps in the degradation of biologically active GA appear to involve hydroxylation reactions that are similar to the final steps of GA synthesis. Similarly to the GA3-oxidases, which hydroxylate the 3-position of GA precursors to form active GAs (Chiang et al., 1995; Talon et al., 1990), GA-2 oxidases hydroxylate the 2-position of active C19-GAs as a first step to render them inactive (Sakamoto et al., 2001; Martin et al., 1999; Thomas et al., 1999). Two additional GA2-oxidases (AtGAox7 and AtGAox8) have recently been isolated from Arabidopsis that hydroxylate C20-GA precursors, but not active C19GAs (Schomburg et al., 2003in press). When over-expressed in transgenic Arabidopsis and tobacco, these genes result in plants with decreased levels of active GAs and a range of corresponding dwarf phenotypes. Transgenic CaMV35S::AtGAox7 and AtGAox8 petunias have a range of dwarf phenotypes, suggesting that the control of endogenous GA levels may be applicable to important floriculture crops as well (Figure 45.1). It is interesting to note that the dwarf phenotype can be reversed by treatment of plants with exogenous GA (Figure 45.2). This is very important for the application of dwarfing technology to the ornamental plant industry, because producers of vegetative propagules may actually want to temporarily increase shoot elongation at various points during cutting production, then have the plants resume a dwarf phenotype after propagation.Breeder selection for the appropriate plant height among transgenic lines will be critical for applying these technologies to any given crop, and breeders should be able to select any particular plant height they desire. Traditional horticultural experiments similar to those used in any breeding programme with a focus directed towards plant height control should be relatively straightforward, and are well established in the industry.

It is likely that dwarfing technologies will find utility in many areas of ornamental plant production. There should be ample room in the industry for potted plants that fit in transit vessels easily, annual bedding plants that do not grow out of the confines of a small garden spot, and shrubs and trees that require less pruning. One extremely important application for dwarfing technology will likely be observed in plant species used in the turfgrass industry. Slower growing lawn grasses that require significantly less labour input for maintenance have been a dream of suburban homeowners and turfgrass maintenance experts for many years. This concept is being developed by commercial scientists at the Scotts Company, with a great deal of progress being made in producing dwarf creeping bentgrass (Agrostis sp.). In high-management areas, these ‘low mow’ grasses have a great deal of potential for saving money on labour costs and equipment maintenance. In addition, slow-growing grasses could help reduce lawn mower pollution and use of fossil fuels, and may use less water and fertilisers. It is likely that these ‘low-mow’ turfgrasses will be important for homeowners and municipal or roadside situations where there is a low amount of foot traffic. It is not likely that these grasses will be used to any great extent on golf courses or sports turf facilities. Due to the high amount of wear and tear that is experienced under these conditions,they may not grow fast enough to cover the damage inflicted by constant use.

Another aspect of hormonal control of plant morphology currently being pursued in ornamental crops concerns the engineering of plants with delayed leaf senescence. Senescence is the final developmental process in the lifecycle of a leaf through which macromolecules (e.g. chlorophyll, proteins, nucleic acids) of leaf cells are metabolised to basic components and transported to the growing shoot and reproductive organs of the plant (Matile et al., 1996; Noodén et al., 1997). Natural leaf senescence in many plants is characterised by lower leaf yellowing or chlorosis as nutrients and other components of the cells are degraded (especially chlorophyll). In horticultural terms, leaf chlorosis can cause a decreased aesthetic appearance of ornamental plants and thus a decrease in the salability of those plants.

One way to prevent leaf senescence is through the manipulation of cytokinins. Cytokinins are an important class of phytohormones that influence numerous aspects of plant growth and development, and have been shown to delay and, in some cases, reverse the leaf senescence process (Gan and Amasino, 1996). The limitation of the use of cytokinins in the prevention of senescence has most often been related to difficulties of controlling temporal and spatial delivery of cytokinins. Exogenously applied cytokinins may not enter the cells, nor be transported to the area needed, nor quickly become conjugated or metabolised to non-active forms (Klee and Lanahan, 1995; Gan and Amasino, 1996; Kaminek et al., 1997). Researchers have recently turned to the manipulation and engineering of endogenous cytokinin biosynthesis in order to solve these problems.

After many years of research on cytokinins, the mechanisms of biosynthesis and perception are just now being elucidated. One enzyme, isopentenyl transferase (IPT), has received much research attention because of its known involvement in cytokinin synthesis. IPT enzyme activity has been isolated from many organisms including plants (Nishinari and Syono, 1980; Blackwell and Horgan, 1994), Dictyostelium discoideum (Taya et al., 1978; Ihara et al., 1984), and Rhodococcus fasciens (Crespi et al., 1992). Although it is well known that there is IPT activity in plants, the genes that encode IPT have only recently been isolated and cloned from plants (Kakimoto, 2001; Takei et al., 2001). As a result, experiments focused on the manipulation of endogenous cytokinin synthesis in transgenic plants with these genes have been lacking. In contrast, a gene from Agrobacterium tumefaciens that encodes the IPT enzyme has been available for use in transgenic plant research for a number of years. IPT is encoded on the Ti (tumour inducing) plasmid of Agrobacterium tumefaciens (Akiyoshi et al., 1984; Barry et al., 1984). It catalyses the condensation of dimethylallylpyrophosphate (DMAPP) and 5′AMP to form isopentenyladenosine 5′-phostphate ([9R-5′P]iP), which is then quickly converted to different cytokinins. This is presumed to be the rate-limiting step in cytokinin biosynthesis since expression of this one gene can cause an over-production of cytokinins (Medford et al., 1989; Klee and Lanahan, 1995; Gan and Amasino, 1996). Attempts have been made to use this gene under the control of various promoters, for example, modified CaMV 35S (Faiss et al., 1997), Cu2+-inducible (McKenzie et al., 1998), or heat shock inducible (Smart et al., 1991) to stop leaf senescence. However, the results of such experiments were often complicated by abnormal growth patterns, or the possibility that the induction treatment (i.e. high temperature or CuSO4 treatment) was causing the observed phenotype (Smart et al., 1991; Buchanan-Wollaston, 1997).

In an attempt to overcome the problems associated with past work on transgenic plants overproducing cytokinins, Gan and Amasino (1995) developed a genetic construct that utilised the highly senescence-specific promoter from SAG 12 (PSAG-12) to drive IPT expression. This construct had three important features due to PSAG-12 specificity: temporal regulation, spatial regulation and quantitative regulation (Gan and Amasino, 1995, 1996). When leaf senescence is triggered, the transcription of IPT is activated by PSAG-12, leading to the production of functional IPT enzyme. The enzyme then catalyses cytokinin production, which in turn delays senescence. Without senescence signals, the PSAG-12 promoter attenuates IPT transcription and subsequent enzyme production, thus providing autoregulatory control of cytokinin synthesis. The PSAG-12-IPT construct was transformed into tobacco. The transformed plants displayed a normal growth habit except that senescence was inhibited and there was a significant increase in flower number, an increased biomass due to the presence of lower leaves, and increased seed yield (Gan and Amasino, 1995). Since then, transgenic PSAG-12-IPT lettuce plants (McCabe et al., 2001) Nicotiana alata (Schroeder et al., 2001) and petunias (Clark et al., in press) with similar delayed leaf senescence phenotypes have been produced (Figure 45.3). The PSAG-12 promoter has also been used to drive the expression of the KNOTTED-1 mutant gene from maize in transgenic tobacco plants to confer a delayed leaf senescence phenotype (Ori et al., 1999), thus extending the utility of delayed leaf senescence technologies. Transgenic PSAG-12:KNOTTED-1 and PSAG-13-IPT petunias both have delayed leaf senescence after nutrition stress induced at the onset of flowering. It is likely that horticultural performance studies will show that breeder selection of transgenic plants under a wide range of selection criteria will be essential for providing the growers with the methods required to produce these plants effectively on a commercial scale.
One hormone of particular interest in the ornamental plant industry is ethylene gas. Ethylene is involved in many physiological processes in plants including fruit ripening, petal senescence, abscission and seed germination. Floral senescence is of particular interest because quality loss of many important floriculture crops is known to occur due to ethylene gas in the post-harvest environment. Treatment of ethylene-sensitive flowers with chemical ethylene biosynthesis or perception inhibitors delays visual symptoms of corolla senescence (Jones and Woodson, 1997; Whitehead et al., 1984; Serek et al., 1995). Many economically important crops are grown for their floral display; therefore the control of ethylene synthesis and perception is thought to be the key to increasing display life and enhancing visual quality. Since ethylene is such a significant problem in both potted flowering crops and cut flowers there have been several attempts to produce chemical control methods for both ethylene synthesis and sensitivity. Effective control of ethylene synthesis using a chemical approach has been much less effective across the industry than the manipulation of ethylene sensitivity. Ethylene sensitivity has long been managed in floriculture crops through the use of silver thiosulfate (STS), which makes plant tissue insensitive to ethylene, but also has environmental downsides that appear to restrict its commercial use. Another chemical approach gaining popularity within the industry is the use of 1-MCP (1-methylcyclopropene), which is a compound that blocks the ethylene receptor protein and makes plant tissue insensitive to ethylene. Typically applied as a gas, 1-MCP provides a means by which large amounts of tissue can be treated for a short time. Although this compound has proved to be effective in some crops, there is some difficulty with the use of 1-MCP in crops that continue to produce new ethylene receptor proteins through development during post-harvest transit, thus limiting the residual effect of the compound (Cameron and Reid, 2001).

A great deal of research has been conducted to produce floriculture crops that synthesise less ethylene, or are insensitive to ethylene. Transformation with an antisense ACC oxidase has been shown to be effective in producing carnation (Savin et al., 1995) and torenia plants (Aida et al., 1998) that produce significantly less ethylene, and have delayed flower senescence. As has been the case with chemical approaches to control the effects of ethylene in ornamental crops, the method of control receiving the most research attention in terms of genetic engineering has been at the level of ethylene perception. Wilkinson et al. (1997) transformed petunia with a dominant mutant Arabidopsis ethylene receptor, etr1-1, under the control of a constitutive cauliflower mosaic virus 35S (CaMV35S) promoter to produce ethylene insensitivity throughout the whole plant (Figure 45.4). These petunias had an increase in natural and pollination-induced flower longevity compared to wild-type plants, but had physiological side effects that limit their commercial use (Wilkinson et al., 1997; Gubrium et al., 2000). Ethylene-insensitive petunias showed a significant reduction in adventitious root formation, and even exogenous treatments with auxin did not increase adventitious root formation to untreated wild-type controls (Clark et al., 1999). Since many horticultural species are often propagated through vegetative cuttings this characteristic would severely limit the commercial utility of ethylene-insensitive plants propagated by vegetative cuttings. After much experimental effort, it is clear that the key to manipulation of ethylene sensitivity will be the promoters being used to drive transcription of these proven transgenes. Effective temporal and spatial control of transgene expression will ultimately lead to plants that have longer lasting flowers with no negative side effects that would limit commercial production. Bovy et al. (1999) have supported this idea by producing transgenic ethylene-insensitive carnations by driving the etr1-1 transgene with the flower-specific transcriptional promoter fbp1 from petunia. Although they were able to produce plants with a delayed flower senescence phenotype, they did not report heritability of the trait or extensive investigation of other horticultural performance characteristics that would limit commercialisation of these plants. Future experiments directed towards discovering the appropriate promoter to drive transgenes conferring ethylene insensitivity will likely be the key to success for the development of long-lasting flowers in the future. It will be imperative to conduct a significant amount of field and greenhouse trailing of these plants in order to completely eliminate the possibility of negative side effects.

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