The mitochondrion serves essential functions as the centre of energy metabolism in developing eukaryotic organisms. Pollen development in plants appears to be particularly influenced by mitochondrial function. Rearrangements of mitochondrial DNA that lead to unique chimeric genes sometimes result in an inability of the plant to produce fertile pollen (Fig. 14.1). This process, known as cytoplasmic male sterility (CMS), is particularly useful for the production of hybrid varieties for increased crop productivity and has been extensively reviewed (Schnable and Wise 1998; Kempken and Pring 1999; Linke and Borner 2005; Chase 2007). The association of CMS with abnormal mitochondrial gene expression has been established in many plant species, including maize (Levings 1990), petunia (Bino 1985), and sorghum (Pring et al. 1995; Xu et al. 1995a). It is thought that a disruption in pollen development is a consequence of mitochondrial dysfunction resulting from chimeric genes. The incorporation of the derived proteins into the mitochondrial membrane or multi-protein enzyme complexes may lead to the impairment of mitochondrial function. However, it has only been possible in a few cases to artificially introduce CMS by expressing CMS-associated chimeric genes, thus proving them to be causative agents of CMS (Hernould et al. 1993b; Gomez-Casati
et al. 2002), because these attempts often fail (Stockmeyer et al. 2007). However, there are ways to engineer CMS, e.g. expression of the beta-ketothiolase from Acinetobacter in tobacco plastids conditions maternally inherited male sterility (Chase 2006; Pelletier and Budar 2007). A unique feature of CMS is that the expression of the trait is influenced by nuclear fertility restorer genes (Schnable and Wise 1998; Kempken and Pring 1999). Nuclear restorer genes can suppress the effect of the sterile cytoplasm and restore fertility in the next generation. A number of restorer genes have been shown to encode pentatricopeptide repeat (PPR) proteins (Brown et al. 2003; Desloire et al. 2003; Kazama and Toriyama 2003; Akagi et al. 2004; Wang et al. 2006). The PPR proteins are a large family of 500-600 members in higher plants (Small and Peeters 2000).
Cytoplasmic male sterility has been utilized in some important crops, such as sunflower, rice (Chap. 22), oilseed rape (Chap. 21), and sorghum, to prevent unwanted pollinations, but CMS mutants and restorer systems are not available for all agricultural crops. In some cases, CMS has been associated with increased disease susceptibility. For example, the susceptibility of T-cytoplasm in maize to race T of the southern corn leaf blight (Bipolaris maydis) led to an epidemic in the United States in 1970 (Wise et al. 1987). Cytoplasmic male sterility is only transmitted maternally and all progeny are sterile. These CMS lines must be maintained by repeated crossings to a sister line, the maintainer line, which is genetically identical except for possessing normal cytoplasm and is male-fertile. The main-tainer thus carries the recessive restorer alleles. Fertility restoration is essential in crops, such as corn or sunflower, where seeds are harvested.
Anther and pollen development and fertilization processes have been the subjects of much investigation (Goldberg et al. 1993). Many nuclear genes involved in pollen development have been identified as mutants that lead to pollen abortion and male sterility. This nuclear, or genic, male sterility is useful for hybrid seed production, but it has limitations because female parental lines are heterozygous and their offspring segregate into fertile and sterile plants in a 1:1 ratio. Nuclear male sterility in plants includes both spontaneous natural and engineered sterility. Spontaneous mutations leading to nuclear male sterility commonly occur at a high frequency. Such mutations can be easily induced by chemical mutagens or ionising radiation. Nuclear male sterility is usually controlled by a pair of recessive genes. Generally, these recessive mutations affect a large number of functions and proteins that are, for example, involved in male meiosis (Glover et al. 1998). In many crops, nuclear male sterility does not permit the effective production of a population with 100% male-sterile plants. This fact seriously limits its use in hybrid seed production (see also Chap. 6).
Many different strategies have been reported for the production of male-sterile plants by interfering with the development and metabolism of the tapetum (van de Meer et al. 1992; Hernould et al. 1998) or pollen (Worrall et al. 1992) in transgenic plants since the first transgenic male sterility system was described. Male sterility is further induced by using sense or antisense suppression to inhibit essential genes (Xu et al. 1995b; Luo et al. 2000) or by expressing aberrant mitochondrial gene products (Hernould et al. 1993a; He et al. 1996; Gomez-Casati et al. 2002). However, all of the available strategies have drawbacks, such as metabolic or general development interference or being restricted to specific species. Thus, a universal and dominant male sterility system with efficient effects on pollen growth, and offering the possibility to efficiently restore fertility, would be a great advantage for the production of hybrid seeds.
14.3.1 The Selective Destruction of Tissues Important for the Production of Functional Pollen
One way to achieve male sterility systems, is the use of a gene which encodes a protein that is able to disrupt cell function, for example a ribonuclease that destroys the RNA of the tapetal cells (Mariani et al. 1990; Mariani et al. 1992; Burgess et al. 2002). A well known example of this kind is the Barnase/Barstar system shown in Fig. 14.2. Using the PsEND1 promoter is a novel method of producing genetically engineered male-sterile plants by early anther ablation (Roque et al. 2007). The PsEND1 promoter belongs to an anther-specific gene from pea that confers very early gene expression in anther primordium cells. The authors fused this promoter to the barnase gene.
Wild-type fertile plant i
Normal tapetum development
Fig. 14.2 The Bamase/Barstar system. (a) Normal tapetum development in the wild-type plant. (b) Tapetal-specific promoter Ta29 drives expression of the barnase gene, leading to male-sterile plants. (c) Barnase inactivated by barstar inhibitor, resulting in restored male fertility. Based on data from Mariani et al. (1990, 1992)
Another way to introduce male sterility is the use of diphtheria toxin A-chain (Koltunow et al. 1990), which is expressed in a tissue-specific manner. The tapetum serves as a good target for these expression strategies because it plays a critical secretion role in the process of pollen formation. In some of these systems, sterility or fertility can be chemically regulated. For example, inducible sterility can be obtained through the expression of a gene encoding a protein that catalyses the conversion of a pro-herbicide into a toxic herbicide only in male reproductive tissues. In transgenic Nicotiana tabacum plants, male sterility was introduced by tapetum-specific deacetylation of the externally applied non-toxic compound N-acetyl-L-phosphinothricin (N-ac-Pt) (Kriete et al. 1996). Transgenic tobacco plants expressing argE from Escherichia coli under the control of the tapetum-specific tobacco TA29 promoter were produced. The gene product of argE represents an N-acetyl-L-ornithine deacetylase, which removes the acetyl group from N-ac-Pt, resulting in the cytotoxic compound L-phosphinothricin (Pt, glufosinate). The application of N-ac-Pt leads to empty anthers, resulting in male-sterile plants. Another example of tissue-specific cell ablation is the use of a bacterial phosphonate monoester hydrolase as a conditional lethal gene (Dotson et al. 1996).
In Arabidopsis thaliana, pehA from Burkholderia caryophilli, a glyphosate metabolizing bacterium, has been expressed using a tapetum-specific promoter. The treatment of transgenic plants with the protoxin glyceryl phosphate leads to male sterility because of the hydrolysis to glyphosate, a potent herbicide inhibiting the biosynthesis of aromatic amino acids. Another example for such
Restored male-fertile plant
|Ta29| barnase > |Ta29| barnase > |Ta29 barsta
) Tapetal-specific Barstar
Destruction of RNA in tapetum
Barnase inactive chemical control is the inducible expression of a male-sterility gene by the application of a chemical (Mariani et al. 1990; Goff et al. 1999). In order to induce fertility, the expression of a fertility restorer gene that can complement the sterility, or a male sterility gene repressor, can be chemically controlled (Cigan and Albertsen 2000).
An alternative method for fertility restoration has been suggested by Luo et al. (2000). They used a site-specific recombination system, FLP/FRT from yeast, to restore fertility in Arabidopsis plants that were male-sterile due to the antisense expression of the pollen- and tapetum-specific bcpl (Mariani et al. 1992) restored the fertility of male-sterile plants generated through the use of the bacterial extracellular ribonuclease Barnase (Paddon et al. 1989) by expressing a specific inhibitor of Barnase, called Barstar (see Fig. 14.2).
Ethylene controls many physiological and developmental processes in plants, including fruit and flower development. Ethylene exerts its effects through the ethylene receptor, which has been isolated in a variety of plant species. The overexpression of mutated melon ethylene receptor genes affects pollen development and induces a male-sterile phenotype in transgenic plants. The inducible male sterility system using mutated ethylene receptor genes could be a possible strategy for preventing pollen dispersal from these plants, thereby reducing the potential impact associated with transgenic plants. The system has been tested in tobacco and lettuce (Lactuca sativa; Takada et al. 2005; Ma et al. 2006; Takada et al. 2006; Takada et al. 2007).
Yet another, though quite unusual, approach is based on the nuclear expression of the mitochondrial atp9 from wheat (see also Sect. 14.3.3). In plant mitochondria, the atp9 transcript is subject to RNA editing. This editing process is believed to be essential for the function of the encoded peptide. To obtain male-sterile plants, the unedited sequence is fused to a mitochondrial targeting sequence and expressed under control of three different promoters in A. thaliana. Indeed male-sterile plants have been obtained (Hernould et al. 1993b; Gomez-Casati et al. 2002).
14.3.2 Changing the Levels of Metabolites Needed for the Production of Viable Pollen
Another approach to induce male sterility in plants is the metabolic engineering of the carbohydrate supply. Carbohydrates are important for anther and pollen development. The extracellular invertase Nin88 mediates the phloem unloading of carbohydrates via an apoplastic pathway. Tissue-specific antisense repression of nin88 in tobacco causes male sterility because early stages of pollen development are blocked (Goetz et al. 2001). McConn and Browse (1996) demonstrated that Arabidopsis triple mutants that contained negligible levels of trienoic fatty acids, such as jasmonate, were male-sterile and produced no seed. In that case, the fertility could be restored through the exogenous application of jasmonate.
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