ELP6

Tweaks to the mitochondrial genome hold the potential for better hybrid seed production or to introduce seedless fruits. North Carolina State University researchers have successfully transferred an important gene from one compartment of a plant cell to another to produce tobacco plants that lack pollen and viable seeds, while otherwise growing normally. Their findings could lead to better ways of producing hybrid seeds to maximize crop productivity, or to introduce seedlessness in fruit species lacking the often-desired trait, such as raspberries, blackberries or muscadine grapes. The researchers began the work in the energy-producing portion of a cell, the mitochondria. In plants, aberrations within the mitochondrial genome can be associated with the inability to make pollen, a trait known as cytoplasmic male sterility (CMS) that has been successfully exploited for the production of high-yielding hybrid seeds in many important crops. Naturally occurring CMS-based systems that are robust enough to facilitate commercial scale hybrid seed production are limited, however. In their proof-of-concept study, the NC State researchers, together with colleagues from Precision BioSciences and Elo Life Systems, deployed a unique strategy to test whether the CMS trait could be generated in tobacco, a commonly used model species in plant research. The researchers initially took an essential mitochondrial gene called atp1 and moved it to the nucleus after placing it under the regulatory control of an element -- known as a promoter -- that they predicted would allow the transferred atp1 gene to be expressed in every cell of the plant except those responsible for producing pollen. The researchers then used genome editing tools to permanently remove the native atp1 gene from the mitochondria. Their approach was successful. "The results exceeded our expectations," said Ralph Dewey, Philip Morris Professor of Crop Science at NC State and corresponding author of a paper describing the research. "The plants looked completely normal up until they began to flower, but then failed to produce pollen because the transferred atp1 gene was no longer expressed. Importantly, because the original atp1 gene was deleted from the mitochondrial genome, the trait will be inherited maternally, which is a crucial consideration for large-scale hybrid seed production." Pollen wasn't the only casualty of the technique. When cross-fertilized using pollen from a neighboring normal plant, their tobacco plants unexpectedly produced small, hollow seeds, much like those seen in popular "seedless" fruits such as watermelons and grapes. "That's because the promoter that we chose not only failed to express during pollen formation, but also during early seed development," Dewey said. Dewey said that his team is now working to decouple these results so that researchers can achieve either pollen infertility or the seedless trait alone, instead of both at the same time. Dewey also stressed that the findings shouldn't be limited to tobacco plants. Their next generation of experiments will include testing the seedless trait in tomato, a close relative of tobacco. They will also test their novel CMS trait in a grain such as rice to test the efficacy of their system in a crop where hybrid seed production is important for achieving maximal yields. "Knowing the way the system works, there's no reason to believe that we couldn't effectively transfer the technology to other plant species," he said. The study appears in Frontiers in Plant Science. Devarshi Selote, H. Carol Griffin, Allison N. Dickey, Derek Jantz, J. Jeff Smith, Anna Matthiadis, Josh Strable, Caitlin Kestell and William A. Smith co-authored the paper. The work was funded in part by a grant from Elo Life Systems and supported by the NC State Plant Breeding Consortium. Dewey and several other researchers have filed a patent on the new technique.

Biologists demonstrate how the auxiliary factor CGL160 contributes to the synthesis of crucial parts of the photosynthetic machinery. LMU biologists demonstrate how the auxiliary factor CGL160 contributes to the synthesis of crucial parts of the photosynthetic machinery. Photosynthesis is the means by which plants, algae, and cyanobacteria extract their 'food' in the form of energy-rich biomolecules from sunlight, carbon dioxide, and water. It is a complex process, from which researchers are still coaxing many new details. A team led by LMU biologists Thilo Rühle, Bennet Reiter, and Prof. Dario Leister has now solved another piece of the puzzle of this essential process and elucidated the role of the auxiliary factor CGL160, as the scientists report in the journal The Plant Cell. Photosynthesis takes place in several partial reactions at the so-called thylakoid membranes in the chloroplasts, where various pigments absorb the electromagnetic radiation of sunlight. Specific protein complexes in conjunction with ATP synthases then convert this light energy into chemical energy in the form of ATP. This enables the plant, among other things, to synthesize carbohydrates as energy-rich 'fuel' for cellular respiration. As 'molecular machines,' ATP synthases are therefore a vital component of plant metabolism. How ATP synthases are assembled in the cell has not yet been fully clarified. Based on the model organism Arabidopsis thaliana, the researchers have now been able to demonstrate that the protein CGL160 plays a key role in the process by recruiting the coupling factor CF1 of the ATP synthase. "The protein CGL160 sits with its base in the thylakoid membrane, while its N-terminal domain protrudes like a rod and fishes the soluble CF1 portion of the ATP synthases out of the fluid inside the chloroplasts. This portion of the protein binds the CF1 'headpiece' and facilitates linkage with the portion of the ATP synthase embedded in the thylakoid membrane, making its formation considerably more efficient," explains Thilo Rühle. Evolutionary roots and adaptations Even under unfavorable growth conditions, the presence of auxiliary factors such as CGL160 prove to be advantageous: The LMU biologists discovered that an absence of CGL160, with its CF1-binding function, under conditions of light deficiency negatively impacts the development of chloroplasts. "We observed that the chloroplast structure of corresponding mutants suffers more during short days with just eight hours of daylight. We advance the hypothesis therefore that the interaction between the N-terminal domain of CGL160 and CF1 is also an evolutionary adaptation that enables plants to cope better in various light conditions," says Rühle. The researchers also found clues as to the evolutionary origin of CGL160. In experiments with Arabidopsis thaliana, they succeeded in functionally replacing the membrane domain of CGL160 with the Atp1 protein from cyanobacteria of the genus Synechocystis. "Around 80 percent of the function can be restored in this way. This suggests that CGL160 in land plants may have come from a predecessor protein of cyanobacteria," says Rühle. Indeed, plant cells acquired their chloroplasts through incorporation and functional integration of cyanobacteria. Potential route to regulation of photosynthesis This set in train the emergence of a complex synthesis plan for photosynthetic machinery. To better understand this plan, the LMU biologists now want to identify all auxiliary factors -- in addition to CGL160 and a handful of other currently known factors -- that are important for the assembly and function of ATP synthases in plant thylakoid membranes. "We assume that there are much fewer factors involved here than in the photosystems themselves -- accordingly, ATP synthase is an easier potential route for the regulation of photosynthesis," says Rühle. "In the future, people could use this knowledge for various applications, such as modifying plants so that they can optimally cover their ATP requirements even when exposed to stressors like heat or extreme light conditions."