Discovery of a plant hormone that affects other organisms

Shinjiro Yamaguchi
Team Leader
Cellular Growth and Development Research Team
RIKEN Plant Science Center
High-school biology textbooks describe auxin, cytokinin, gibberellin, abscisic acid and ethylene as plant hormones. Now, a new hormone, strigolactone, is joining the group. Strigolactone was discovered more than 40 years ago to be involved in promoting germination in root-parasitic plants. Shinjiro Yamaguchi, team leader of the Cellular Growth and Development Research Team at the RIKEN Plant Science Center, has recently clarified that strigolactone, which is also known for its role in attracting mycorrhizal fungi, is in fact a plant hormone that inhibits shoot branching. This is the first discovery of a plant hormone that affects external organisms. These findings are expected to encourage the development of new methods for controlling root-parasitic plants, which are responsible for serious damage to agricultural crops in Africa. Ultimately, the discovery is expected to lead to improved crop quality and yield.

What are plant hormones?

In August 2008, news agencies around the world reported the “Discovery of a Plant Hormone that Inhibits Shoot Branching”. As Yamaguchi appreciates, “this was an important discovery that should be added to textbooks.”

What are plant hormones? “Plant hormones are low molecular weight compounds produced by plants themselves. They control the germination, growth and response of plants to their environment in minute amounts. The same hormones are produced by many different types of plants,” explains Yamaguchi. In contrast to the hormones found in animals, which have a specific function at a certain location and for a certain time, plant hormones have diverse functions at various times and locations.

Since receiving his PhD from the University of Tokyo, Yamaguchi has been heavily involved in research on plant hormones, particularly gibberellins. Gibberellins were discovered by Eiichi Kurosawa in 1926 and were further investigated by Teijiro Yabuta, a former chief scientist at RIKEN. Today, it is known that gibberellins promote plant growth and are related to the induction of seed germination and plant height. Yamaguchi has achieved many breakthroughs in his research on gibberellins, including identification of the genes of enzymes responsible for gibberellin production, elucidation of a gibberellin-induced control mechanism for the germination of seeds, and the discovery of a molecular mechanism that deactivates gibberellins. However, there remains a key question that he has been attempting to answer for a long time. “So far, we’ve discovered several plant hormones, but is that really all of them? Are there any other plant hormones that we don’t know about yet?”

Focusing on mutant plants with excess shoot branching

Yamaguchi had one particular mutant plant in mind—a pea mutant with excess shoot branching. It was discovered by an Australian research group in the mid-1990s.

“A branch is an adult form of axillary bud, which emerges laterally from the base of the leaf stem. However, not all axillary buds grow to become adult branches. Plants have a mechanism called apical dominance, by which axillary buds are prevented from growing at the same time as the terminal bud—the tip of the plant stem. Cytokinin and auxin are the plant hormones that control apical dominance; cytokinin promotes the growth of axillary buds, while auxin inhibits growth. It was believed that the growth and shoot branching depend on the balance between cytokinin and auxin.”

In the mid-1990s, a group of Australian researchers investigated the amount of cytokinin and auxin contained in a mutant pea plant with excess shoot branching. They presumed that since the axillary buds in the dormant state started to grow with repeated branching, there must be an excess accumulation of cytokinin that promotes the growth of axillary buds. “Their results showed the opposite,” says Yamaguchi. There were smaller amounts of cytokinin and larger amounts of auxin. “The functions of auxin and cytokinin could not always explain the excess shoot branching of the mutant plant. It was also known that shoot branching returned to the normal state when the mutant plant was grafted onto a normal pea plant. These facts made me think that there must be a plant hormone, other than auxin, that is capable of inhibiting shoot branching—a branching-inhibiting hormone that this did not function in the mutant, resulting in excess shoot branching.”

Since then, mutants with excess shoot branching have been observed in other plants, including petunia (a popular garden plant), Arabidopsis thaliana (commonly used in experiments) and even rice plants. “It is highly possible that a plant hormone is involved in excess shoot branching because the same phenomenon has been observed in various mutant plants. I was convinced that studying these mutant plants would lead to the discovery of a previously unknown plant hormone,” says Yamaguchi.

Discovery of a new plant hormone

To identify the new plant hormone, Yamaguchi began research on branching-inhibiting hormones in 2005 using mutant rice plants with excess shoot branching. Three mutant rice plants were known to exhibit excess shoot branching, each with respective deficiencies of D17, D10 and D3 genes.

Genes D17 and D10 were shown to produce carotenoid cleavage dioxygenase (CCD), which cleaves a group of pigments called carotenoids. “This suggested that the branching-inhibiting hormone was produced when the carotenoids were cleaved. The D17- and D10-deficient mutant plants exhibited excess shoot branching because no branching-inhibiting hormone was being produced. Gene D3 was considered to be a receptor for the shoot-branching hormone, or to be the gene responsible for the production of signal-transmitting proteins. It was concluded that the D3-deficient mutant plant produced branching-inhibiting hormones, but failed to respond to them, leading to excess shoot branching.”

To identify the branching-inhibiting hormone, Yamaguchi conducted a series of bioassays to explore the response of the mutant plants to various compounds. “We wanted to find a compound that returns the D17- and D10-deficient mutant plants, which do not produce the branching-inhibiting hormone, to the normal branching state but does not affect the D3-deficient mutant, which produces the hormone but does not respond to it.” Based on this idea, Yamaguchi administered the mutant plants with a liquid extracted from the tissue of plants that were believed to contain the branching-inhibiting hormone. If the extract elicited the expected response, the components of the extract were isolated and administered separately. This cycle of separation, administration and examination was repeated many times until the target compound was eventually identified.

The bioassay technique is a standard approach to identifying a target compound. However, Yamaguchi had a hard time narrowing down the target compound because of the large number of compounds produced by plants. The stalemate was finally broken in October 2005 when a paper on the root-parasitic plant Striga was published.

Striga is found in dry regions of Africa and South Asia, and is a parasite plant that attacks the roots of monocotyledonous plants, such as corn and sorghum, and absorbs nutrients and water from the host plant to grow. Striga attacks inhibit normal growth of the host plant, and are responsible for massive reductions in crop yields, posing a serious problem for agriculturalists.

The most striking feature of Striga is that its seeds germinate only when they recognize the presence of the compound called strigolactone, which is secreted from the roots of host plants. The secretion of strigolactone has been known for 40 years, but the question of how strigolactone is produced had remained a mystery. The 2005 paper showed that strigolactone is produced from carotenoids, much like the branching-inhibiting hormone that Yamaguchi was looking for, which is produced when carotenoids are cleaved by CCD. “There are many kinds of CCD. However, the investigation showed that there were only a few CCDs for which the functions had not been determined. Thus, I formed the hypothesis that strigolactone is the branching-inhibiting hormone we were looking for.”

Yamaguchi then began conducting experiments to verify his hypothesis. He found that almost no strigolactone was produced in D17- or D10-deficient mutant plants, whereas large amounts of strigolactone were produced in D3-deficient mutant plants. “I felt sure that my hypothesis was correct. Based on previous studies, we knew that mutant plants that do not respond to gibberellin produce around 100 times the normal amount of gibberellin. Thus, the phenomenon could be reasonably explained if the D3-deficient mutant plants are plants that do not respond to strigolactone.”

When strigolactone was administered to mutant plants, Yamaguchi was able to verify that the excess shoot branching observed in the D17- and D10-deficient mutant plants returned to normal, while the D3-deficient mutant plants were unaffected. “There is now no doubt that strigolactone is a branching-inhibiting hormone.” Strigolactone was thus confirmed to be a newly discovered plant hormone that inhibits shoot branching.

Yamaguchi attributes this textbook-revising breakthrough to two factors. “We had accumulated a great deal of know-how through our research into gibberellin. This is the first point. In addition, we were fortunate enough to be able to use the ultra-sensitive mass spectrometer at the RIKEN Plant Science Center. The amount of plant hormone contained in one gram of plant tissue is only one nanogram. Without the RIKEN mass spectrometer, we could not have achieved the structural analysis using such a small amount of fragile strigolactone.”

Unexpected relationship between branching and mycorrhizal fungi

Despite his success, Yamaguchi felt that something still remained unclear. “Why does a plant secrete a germination-inducing substance that attracts root parasites? This is clearly contrary to the natural survival of the plant.”

A research team at Osaka Prefecture University recently solved this mystery when they clarified that strigolactone attracts mycorrhizal fungi. Whereas Striga absorbs nutrients from the host plant without benefit for the host, mycorrhizal fungi perform a highly beneficial function: in return for invading the root cells of a plant and consuming some of the host plant’s photosynthesized sugars, mycorrhizal fungi pass on water and nutrients, such as phosphorus, that the fungi absorb from the soil through their fibrillose hyphae. “Mycorrhizal fungi develop a symbiotic relationship with the host plant, and the host plant actively secretes strigolactone to attract the beneficial mycorrhizal fungi.”

Yamaguchi has yet more questions, such as why a single plant hormone is involved in both branching inhibition and mycorrhizal fungi attraction at the same time. “We think that this is the survival strategy of the host plant against a shortage of nutrients,” says Yamaguchi. “In a nutrient-poor environment, host plants need to attract mycorrhizal fungi, which help the plants absorb nutrients. At the same time, the root needs to prevent the stem from unfruitful shoot branching in a nutrient-poor environment. This information can be transmitted more effectively if a single plant hormone is used than if two separate signal molecules are used.”

Protecting crops from root parasitic plants

Yamaguchi’s findings are expected to lead to the development of a Striga control method. “Many researchers have been searching for plants like the D10-deficient mutants that do not produce strigolactone, because these plants cannot be parasitized by Striga.”

With the cooperation of Ken Shirasu of the Plant Immunity Research Group at the RIKEN Plant Science Center, Yamaguchi has been able to examine whether D10-deficient mutant plants are resistant to Striga parasitism. When Striga seeds were placed around a normal rice plant, 20% of the seeds germinated, and 10% were found to be parasitic. However, when Striga seeds were placed around a D10-deficent mutant, almost no seeds germinated, and no parasitism was observed. “The results were what we had expected, but there are still problems,” says Yamaguchi. “If no strigolactone is produced, more shoot branching will occur, but the plant will also be unable to attract mycorrhizal fungi, which will adversely affect plant growth. Thus we are currently conducting research to find compounds that attract mycorrhizal fungi but not root-parasitic plants. In cooperation with researchers in Africa, we hope to find a way of preventing crop damage caused by root-parasitic plants, thus contributing to solving the African food problem.”

Developing a technique that can control shoot branching would greatly contribute to agriculture and horticulture. The number of branches ultimately affects the number and quality of flowers, fruit and seeds. For example, higher-quality tomatoes could be produced in lower yield. There are many other plants, including Nicotiana tabacum and chrysanthemum, for which crop yields and quality could be modified through branching control.

What obstacles are there to practical use of these findings in agriculture and horticulture? “It may be that strigolactone is converted into another active form when it functions. Thus, it is necessary to elucidate the main part that acts as a plant hormone. In addition, it is important to find strigolactone receptors; if we find differences between the receptors of plants, mycorrhizal fungi and root-parasitic plants, we may be able to attract mycorrhizal fungi without attracting root-parasitic plants,” says Yamaguchi.

“Strigolactone can affect living things other than plants—no other plant hormone has been found to have this function. Although we have a long history of research in plant hormones, we are still ignorant about many things. I think there are other plant hormones that still remain undiscovered, and I really hope to find them.”

About the Researcher

Shinjiro Yamaguchi

Shinjiro Yamaguchi graduated from the Department of Agricultural Chemistry, Faculty of Agriculture, at the University of Tokyo in 1991. He obtained his PhD in 1996 from the same university, and subsequently joined the Frontier Research Program at RIKEN researching the identification of gibberellin biosynthesis genes in plants. He worked at Duke University in the United States as a postdoctoral fellow from 1997 to 2000, where he studied the regulation of gibberellin biosynthesis in Arabidopsis. He worked at the Laboratory for Cellular Growth and Development, RIKEN Plant Science Center from 2000 to 2005, where he studied regulatory mechanisms for seed germination and plant isoprenoid biosynthesis pathways. He has since been studying plant growth hormones as leader of the Cellular Growth and Development Research Team.