ENGINEERING CYTOPLASMIC MALE STERILITY VIA THE CHLOROPLAST GENOME
Henry Daniell
September, 2005

Naturally-occurring cytoplasmic male sterility (CMS) has been known for over 100 years. CMS systems are used to produce commercial F1 hybrid lines. Ruiz and Daniell (2005) recently reported the first engineered cytoplasmic male sterility system in plants. They studied the effect of light regulation of the phaA gene coding for β-ketothiolase engineered via the chloroplast genome. The phaA gene was efficiently expressed in all tissue types examined, including leaves, flowers, and anthers. The transgenic lines were normal except for the male sterile phenotype, lacking pollen. Scanning electron microscopy revealed a collapsed morphology of the pollen grains. Transgenic lines showed an accelerated pattern of anther development, affecting their maturation, and resulted in aberrant tissue patterns. Abnormal thickening of the outer wall, enlarged endothecium, and vacuolation decreased the inner space of the locules, affected pollen grain, and resulted in the irregular shape or collapsed phenotype. Reversibility of the male sterile phenotype was observed under continuous illumination, resulting in viable pollen and a copious amount of seeds. This study offers a new tool for transgene containment for both nuclear and organelle genomes and provides an expedient mechanism for F1 hybrid seed production.

History of Cytoplasmic Male sterility
Male-sterility-inducing cytoplasms have been known for over 100 years. In 1921, Bateson and Gairdner reported that male sterility in flax was inherited from the female parent. Chittenden and Pellow observed in 1927 that male sterility in flax was due to an interaction between the cytoplasm and nucleus. In 1943, Jones and Clarke established that male sterility in onion is conditioned by the interaction of the male-sterile (S) cytoplasm with the homozygous recessive genotype at a single male-fertility restoration locus in the nucleus. The authors also described the technique used today to exploit cytoplasmic-genic male sterility (CMS) for the production of hybrid seeds. CMS inbred lines have been widely used for hybrid production of many crops. The first application of organelle biotechnology was the role played by cytoplasmic male sterility in hybrid seed production, a major contribution towards the "Green Revolution." The use of cytoplasmic male sterility in hybrid seed production has been recently reviewed by Havey (2004).

The use of CMS for hybrid seed production received a "black eye" after the epidemic of Bipolaris maydis on T-cytoplasmic maize. This epidemic is often cited as a classic example of genetic vulnerability of our major crop plants. In addition to Southern corn blight (CMS-T), cold susceptibility (CMS Ogura) and Sorghum Ergot infection in the unfertilized stigma have been reported. However, these disease linkages were successfully broken by somatic cell genetics and conventional plant breeding. Hybrids of other crop plants may be produced using nuclear male sterility. A natural source of nuclear male sterility was identified in leek. Engineered sources of nuclear male sterility have been developed in model systems. A problem with these approaches is that they segregate for male fertility or sterility and must be over-planted and rogued by hand or sprayed with herbicides to remove male-fertile plants. Male-sterility systems have been created by several different mechanisms; most of these affect tapetum and pollen development. Unfortunately, additional severe phenotypic alterations that were due to interference with general metabolism and development had precluded their use in agriculture.

Advantages of the chloroplast transformation system
A chloroplast genetic engineering approach offers a number of attractive advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position effect due to site-specific transgene integration, and lack of pleiotropic effects due to sub-cellular compartmentalization of transgene products. Genetically engineered cytoplasmic male sterility via the chloroplast genome may be used for the safe integration of foreign genes via the nuclear genome and in those rare cases in which plastid genomes are paternally or biparentally transmitted. Recently, plastid transformation was demonstrated in carrot with their ability to grow in very high saline conditions (up to 400 mM sodium chloride). Additionally, plastid transformation of recalcitrant crops such as cotton and soybean allows the application of the cytoplasmic male sterile system to commercially important crops. For a recent review of developments in this field, see Daniell et al. (2005) and Grevich & Daniell (2005).

Novel mechanism of Cytoplasmic Male Sterility
PHB synthesis takes place by the consecutive metabolic action of β-ketothiolase (phaA gene), acetoacetyl-CoA reductase (phaB) and PHB synthase (phaC). Nawrath et al. (1994) introduced the phbA, phbB, and phbC genes in individual nuclear Arabidopsis transgenic lines and reconstructed the entire pathway, targeting all enzymes to the plastids. This approach resulted in PHB expression up to 14% leaf dry weight. This study suggests that the depletion of metabolites from essential metabolic pathways in the cytoplasm might have caused the pleiotropic effects, and that by targeting the enzymes to the chloroplast, which is a compartment with high flux through acetyl-CoA, the adverse effects were overcome. With expression of optimized gene constructs, PHB yield increased up to 40% of leaf dry weight, but this was accompanied by severe growth reduction and chlorosis (Bohmert et al., 2000), indicating that targeting the PHB pathway to the chloroplast can result in pleiotropic effects, at higher concentrations of polymer synthesis. In an attempt to address the role of phaA expression in the pleiotropic effects observed in transgenic plants expressing PHB, Bohmert et al. (2002) expressed the phbA gene via the nuclear genome. Continuous expression of the phbA gene led to a significant decrease in transformation efficiency, inhibiting the recovery of transgenic lines, and prevented analysis of plants expressing the β-ketothiolase gene. Such a toxic effect exerted by phbA expression was speculated to be the result of PHB biosynthesis intermediates or its derivatives, the depletion of the acetyl-CoA pool, or of the interaction of β-ketothiolase with other proteins or substrates.

While investigating the phb operon, Ruiz & Daniell (2005) proposed to address the specific role of β-ketothiolase (phaA gene) that results in pleiotropic effects in transgenic plants expressing the polyhydroxybutyrate pathway. Therefore, they hyper-expressed the β-ketothiolase gene by inserting the transgene into the chloroplast genome under light regulation and assessed the effects of phaA expression in the chloroplast transgenic plants at molecular and physiological levels. In this study, the phaA gene was constitutively expressed, and fully regenerated transgenic plants were recovered. Therefore, evaluation of the specific effect of β-ketothiolase in the transgenic lines was possible for the first time. Detailed characterization revealed that severe pleiotropic effects such as stunted phenotype and chlorosis observed during polyhydroxybutyrate expression were not observed during phaA expression via chloroplast transformation, with the exception of complete (100%) male sterility. Such elimination of pleiotropic effects was not surprising because the chloroplast genetic engineering system has been able to overcome the toxic effects (stunted growth, chlorosis, and infertility) associated with nuclear expression of the trehalose, xylanase, and cholera toxin B subunit genes, producing totally normal plants that were healthy and fertile.

Reversibility of male sterility
To test whether depletion of the acetyl-CoA pool destined for de novo fatty acid biosynthesis in the chloroplast by β-ketothiolase is the cause of the male sterility phenotype, the authors explored if a continuous light could revert male fertility of the chloroplast transgenic lines. Recent reports have shown light-dependent regulation of the key enzyme in fatty acid biosynthesis. Therefore, acetyl-CoA carboxylase (ACCase) male sterile transgenic lines were exposed to continuous light for a period of 10 days. The authors observed that from 20 flowers produced during the 10 days of illumination by the two transgenic plants, four flowers produced pollen, normal length anther filaments, fruit capsules, and seeds. The seeds recovered from the reverted male fertility study were able to germinate in the selection medium, confirming that these seedlings were transgenic and contained the transgene; wild type tobacco seedlings were bleached. These findings support the hypothesis that an increase in ACCase activity outcompetes the removal of acetyl-CoA by β-ketothiolase.

Conclusions
Havey (2004) documents the worldwide use of CMS to produce competitive hybrid cultivars. Major investments of time and resources are required to backcross a male-sterility-inducing cytoplasm into elite lines. These generations of backcrossing could be avoided by transformation of an organellar genome of the elite male-fertile inbred line to produce female inbred lines for hybrid seed production. Because the male-fertile parental and male-sterile transformed lines would be developed from the same inbred line, they should be highly uniform and possess the same nuclear genotype, excluding mutations and residual heterozygosity. Therefore, the male-fertile parental line becomes the maintainer line to seed-propagate the newly transformed male-sterile line. A few generations of seed increases would produce a CMS-maintainer pair for hybrid seed production. An additional advantage of organellar transformation would be the diversification of CMS sources used in commercial hybrid-seed production. Transformation of the chloroplast genome would allow breeders to introduce different male-sterility-inducing factors into superior inbred lines. Introduction of a male-sterility inducing transgene into one of the organellar genomes of a higher plant would be a major breakthrough in the production of male-sterile inbred lines. This technique would be of great potential importance in the production of hybrid crops by avoiding generations of backcrossing, an approach especially advantageous for crop plants with longer generation times. Moreover, transgenes that are engineered into annual crops could be introgressed into wild crops, persist in the environment, and thereby have negative ecological consequences. Therefore, it may be necessary to engineer a male sterility system that is 100% effective.

For vegetable, fruit, or forage crops, restoration of male fertility in the hybrid is not necessary. This simplifies the production of hybrids because effort can be concentrated on maintainer line development, without concern over whether the pollinator restores male fertility in the hybrid. For crops with seeds as the economically important product, such as canola, sunflower, or maize, one or both of the hybridís parents must bring in male-fertility restoration factors or the male-sterile hybrid seed must be blended with male-fertile hybrid seed. In currently available cytoplasmic male sterile lines, the nuclear genome controls various restoration factors and such factors are often located at multiple loci and are poorly understood. However, the authors report restoration of male fertility by changing conditions of illumination. Therefore, this is a novel approach for creating male sterile transgenic plants, which may help advance the field of plant biotechnology through effective transgene containment.

References

Bohmert K et al. (2002) Constitutive expression of the beta-ketothiolase gene in transgenic plants. A major obstacle for obtaining polyhydroxybutyrate-producing plants. Plant Physiol 128, 1282-1290

Bohmert K et al. (2000) Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight. Planta 211, 841-845

H. Daniell, S. Kumar & Duformantel N. (2005) Breakthrough in chloroplast genetic engineering of agronomically important crops. Trends in Biotechnology 23, 238-245

Grevich J & Daniell H (2005) Chloroplast genetic engineering: Recent advances and perspectives. Critical Reviews in Plant Sciences 24, 1-25

Havey MJ (2004) The use of cytoplasmic male sterility for hybrid seed production. In Daniell H, Chase C, eds, Molecular Biology and Biotechnology of Plant Organelles, Springer Publisher: The Netherlands, pp 617-628

Nawrath C, Porier Y, & Somerville C (1994) Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana result in high levels of polymer accumulation. PNAS 91,12760-12764

Ruiz O & Daniell H (2005) Engineering cytoplasmic male sterility via the chloroplast genome. Plant Physiology 138, 1232-1246

Henry Daniell
University of Central Florida
Dept. Molecular Biology & Microbiology
daniell@mail.ucf.edu