Biography of June B. Nasrallah

As Darwin observed more than 100 years ago (1), so-called “self-incompatible” plants are sterile with respect to their own or closely related pollen but can become fertilized and produce seed with pollen from other strains. However, the genetic mechanisms underlying this phenomenon remained an enigma until the 1980s, when June Nasrallah and colleagues at Cornell University (Ithaca, NY) turned their attention to the problem. Using a combination of genetics, cell biology, and molecular analysis, Nasrallah's team demonstrated that recognition of self-related pollen is determined by highly specific interactions between the SRK receptor kinase, which is expressed exclusively in the stigma epidermal cells that capture pollen, and its ligand, SCR, which is expressed exclusively in pollen (2–5).

In recognition of the decades of research leading up to this accomplishment, Nasrallah was elected to the National Academy of Sciences in 2003. Her Inaugural Article, published in this issue of PNAS, describes mutagenesis studies on SCR and interprets how these mutations change the ligand's interaction with its receptor (6). These studies not only provide a basic foundation for plant genetics and evolutionary research, but also have the capacity to transform and improve plant breeding techniques.

A Family Effort

Nasrallah was born and raised June Bowman in Beirut, Lebanon. She developed a taste for science in high school, where the school principal took great care to expose students to the extensive school gardens she had established. “Lots of interesting biology goes on in the flowers,” the principal would reiterate every spring while she conducted tours of the gardens. “Also,” Nasrallah recalls, “biology was one of the few courses where we did hands-on experiments. We actually thought about how biological systems worked, so it was a very different experience from the rest of the curriculum.”

After high school, Nasrallah continued studying biology at the American University in Beirut. The school is considered one of the best in Lebanon, and June was intrigued by the challenge of the rigorous program. During the course of her studies, June met Mikhail Nasrallah, and the two married after June received her undergraduate degree in 1970.

Figure 1.

June B. Nasrallah

While June contemplated where to attend graduate school, Mikhail had completed a doctorate at Cornell University. Despite her qualms about leaving Lebanon, June eventually joined her husband at Cornell. Under the mentorship of geneticist Adrian Srb, she studied the reproductive life phase of Neurospora, a ubiquitous fungal model for genetics study. June took a particular interest in proteins that are turned on during maturation of the mold's fruiting bodies, and she was able to characterize several of these proteins.

During the years June was working on her doctorate, she and Mikhail had a son and a daughter, and June reduced her academic workload by half to accommodate raising children. Nevertheless, she and Srb published five papers together during this period describing the analysis of various Neurospora reproductive proteins (7–11), and she completed her doctoral degree in 1977.

In the meantime, Mikhail had been working on a project of his own examining self-incompatibility in Brassica, a subfamily of cruciferous plants. Broccoli, cauliflower, cabbage, brussels sprouts, and kale are all the same species of Brassica, despite their disparate appearances. “They have just a few gene mutations that give differences in phenotype. They've been bred for very specific characteristics, just like different breeds of dogs have been selected for their specific looks and behaviors,” said June. Like all plants with “perfect” flowers, Brassica flowers have both male parts (the anthers) and female parts (the pistil). Although many flowering species can self-fertilize or produce seed using an individual plant's own pollen, cruciferous plants have a mechanism that prevents self-fertilization or crosses between plants belonging to the same strain.

If pollen grains from one Brassica plant land on the tip of the pistil, or stigma, of a plant belonging to a different strain, the stigma's epidermal cells release water and other compounds, allowing the grains to germinate and develop a pollen tube that carries sperm cells to the ovary. In contrast, if the pollen lands on a plant's own stigma or on a plant from an identical genetic background, the stigma's epidermal cells recognize pollen as being self-related and inhibit the development of pollen tubes. By the 1950s, researchers had recognized that self-incompatibility resulted from genetic variations between strains at a particular site called the S locus (12). However, exactly which genes were involved and how they operated was unknown.

While June finished her doctorate, Mikhail developed several genetic lines of Brassica as part of his doctoral studies and later as a faculty member at the State University of New York in Cortland. In the mid-1960s, he had identified a protein that was tightly linked to the S locus (13). Then, in the early 1980s, June and Mikhail began studying self-incompatibility together. “He had established all the genetic strains we needed and had done a lot of protein work to determine what genes at the S locus might be turned on; he had done all the groundwork. At that time, we knew that we were ready to move on to a molecular analysis of the system,” June said.

Focusing on the protein Mikhail had recently discovered, the couple traced it to a particular gene at the S locus and began efforts to clone that gene. “I had no experience in molecular biology, and neither did my husband,” said June. Fortunately, developmental geneticists Michael Goldberg and Mariana Wolfner, who then were new members of Cornell's faculty, tutored her on molecular biology's various techniques and concepts. Goldberg and Srb also provided her with laboratory space in which to perform her experiments because she held no formal position at a university. In 1985, the Nasrallahs and Goldberg successfully cloned the gene, which they named the S-locus glycoprotein gene (SLG), and published the work in Nature (2). The study was well received, said Nasrallah, because “at the time, there were very few plant genes associated with a complex trait that were cloned.”

The Nasrallahs began searching for university positions where they could work together. Within a year, the pair accepted offers at Cornell and opened a joint laboratory.

Research in Bloom

In 1991, the Nasrallahs and graduate student Joshua Stein cloned a second gene at the S locus (3). Upon examination, the gene's sequence showed that it produced a protein with an extracellular domain, a single pass transmembrane domain, and a cytoplasmic kinase domain, all characteristics of membrane receptor kinases. Although the protein's function was not yet known, June and her collaborators speculated that it was indeed a receptor and named the gene S-locus receptor kinase (SRK). “It was receptor-like, anyway,” she said. “We called it a receptor kinase because it had all the features of a surface receptor.”

Because this putative receptor was expressed only in the stigma epidermis, it made sense to the Nasrallahs to search for its corresponding ligand in pollen. In 1999, working with postdoctoral associate Christel Schopfer, the team located a third gene at the S locus expressed only in pollen and whose protein product was localized in the pollen coat. The gene's sequence was extremely polymorphic, with only tiny portions conserved in most alleles. Because the gene's different variants produced proteins abounding with cysteine, the researchers named the gene “S-locus cysteine-rich” (SCR). To prove that this protein interacted with SRK, the Nasrallah laboratory transferred an SCR allele from one Brassica plant (a “type-A” plant) into a Brassica plant with a different mating type (“type B”). Normally, a type-A plant will accept pollen from a type-B plant, but the researchers found that the pollen carrying the transferred SCR allele was recognized by the type-A plant as being type A. Thus, the SI mechanism kicked in, and the type-A plant rejected the pollen. The researchers published their results in Science (4). “Several groups were searching for self-incompatibility pollen genes, not only in crucifers, but in other self-incompatible species as well. People had been waiting a long time to see what this pollen factor might be,” June said.

The Nasrallahs, together with Schopfer and another postdoctoral associate, Aardra Kachroo, subsequently demonstrated in 2001 that the SCR protein is a ligand for the stigma receptor kinase and binds to it in allele-specific manner (5). In 2002, the Nasrallahs and postdoctoral associate Pei Liu proved that SRK and SCR are the only necessary factors in determining whether a crucifer species is outbreeding or self-fertilizing (14). Working with the well characterized crucifer Arabidopsis, the researchers transferred an SRK/SCR gene pair that they and visiting scientist Makoto Kusaba (15) had isolated from Arabidopsis lyrata, a species that is self-incompatible, into Arabidopsis thaliana, a species that self-fertilizes. The A. thaliana plant became self-incompatible, proving that the two genes are sole determinants of self-incompatibility.

Finicky Affinity

With proof that SRK and SCR function together to promote self-incompatibility, June wondered which regions of these two proteins interacted to prompt the self-incompatibility response. In her Inaugural Article, found on page 911, June and colleagues performed mutagenesis studies on two variants of SCR (SCR6 and SCR13), swapping domains between the two proteins to form several mutant variants (6). By applying these individual variants to Brassica stigmas and then applying pollen, the researchers assayed whether the mutant proteins triggered self-incompatibility by binding with the stigmas' SRK protein.

June's team discovered that by transferring only four amino acids from SCR13 to SCR6 they could switch the latter protein's activity and trigger self-incompatibility in plants with an SRK13 receptor. The researchers further determined that several mutants of the SCR6 and SCR13 proteins did not completely fail to bind with their respective receptors, but instead had varying levels of affinity. “It's more or less, not all or none,” she said. According to the authors, these results may explain how Brassica and other self-incompatible crucifer species naturally developed hundreds of S-locus variants. Through miniscule changes, as tiny as the difference between only a few amino acids in the SCR and SRK genes, each mutant ligand and receptor may have gradually evolved to have matching affinities, thereby triggering self-incompatibility.

Although self-fertilization is convenient for seed development, it is difficult to produce hybrids in self-fertile, agriculturally important species such as tomatoes or rice. Currently, workers produce hybrid seeds in self-fertile plants by removing each plant's anthers by hand, which involves an enormous amount of labor. In the future, breeders may be able to create hybrid seeds in self-fertile species by simply inserting SRK and SCR genes and planting different self-incompatible strains adjacent to each other. “As we've shown in Arabidopsis, you can introduce these two genes and make a plant that has been self-fertile for 5 million years self-incompatible,” June said.

Synergy in Partnership

Future plans for the Nasrallah team include elucidating the signal transduction pathway that leads to pollen rejection and researching the coevolution of SRK and SCR, projects that continue June's stepwise approach to unraveling the complexity of self-incompatibility. And, as they have over the past 25 years, June and Mikhail will continue their division of labor, each focusing on their individual specialties: she will concentrate mostly on molecular biology, and his work will involve mostly genetics. June hinted that this unique partnership could be one reason for the Nasrallahs' continuing success. “It's extremely advantageous having someone so close to talk to about the research,” she said. “It's like a 24-hour immersion in the science.”