Cytokinin for relief from food crises, moss for environmental remediation

The world will reportedly face a food crisis as a result of the population explosion and other factors in the near future. To increase crop yields is a major issue in plant sciences.

At the Biodynamics Research Team in the Plant Productivity Systems Research Group at the RIKEN Plant Science Center, investigations have shown that cytokinin, a plant hormone that activates cell division, is closely associated with rice yields. Team leader Hitoshi Sakakibara, who leads the world in cytokinin research, is working to control the biosynthesis and activation of cytokinin with the aim of increasing the yields of rice, wheat, corn, and other crops. The team is also conducting joint research into mosses that accumulate heavy metals at high concentrations.

From mouse-ear cress to rice

On the top floor of the East Building of the RIKEN Yokohama Institute is a rice culture room with windows admitting natural light. Many rice plants with various mutations, including activation of the plant hormone cytokinin, are grown there. Team leader Sakakibara goes to the culture room many times a week and carefully examines the plants for stem length, leaves, flowers, spikes, and the like. “Merely thinking about the available data is not good enough,” he asserts. “It is important to actually watch and touch the plants, and then think.”

Rice is a special plant for the Japanese, who rely on rice as their staple food. “I think any rice research is meaningless unless it is focused on characteristics that are beneficial to human beings, such as increased disease resistance, higher yields, and better palatability. In our research we emphasize improving crop productivity.” It was five years ago that Sakakibara began his work with rice. Until then, he mainly targeted mouse-ear cress (Arabidopsis thaliana). Why did he switch to rice?

Mouse-ear cress is an annual plant of the rape family. It is commonly used as a model for research in the plant sciences because it can be grown in the laboratory and has a short lifecycle of one to two months, and a low plant height of up to 30 cm. In 2000, its total genome sequence was completed. “In the past 10 years or so, it has been a world trend to use mouse-ear cress to elucidate the basic mechanisms in plants. I began working on this plant in 2000, when I joined RIKEN Plant Science Center.”

Sakakibara soon gained confidence: “The time when we could study just mouse-ear cress was coming to an end. The plant itself is agriculturally useless. I believed that it was time to apply our knowledge about mouse-ear cress to economic plants, and so I gradually expanded my research into rice.” His decision was strengthened by the fact that the complete genome sequence for rice was determined in 2002.

Cytokinin determines rice yields
In June 2005, RIKEN and Nagoya University issued a press release entitled “Key gene for determining rice yields identified.” Sakakibara succeeded, for the first time in the world, in identifying the gene that determines the number of rice grains carried on each spike, and introducing the gene into the famous rice variety Koshihikari to increase its yield. It is a commonly held view that the world will encounter a serious food crisis because of the population explosion and other factors in the near future. Their groundbreaking achievement attracted attention because it may lead to a resolution of the problem.

“So far, many breeders have been engaged in creating new rice plants with higher yields by hybridizing promising cultivars that have useful properties,” says Sakakibara. “However, no one knew why the yields increased. We identified the causal gene, and elucidated the mechanism behind its involvement in the yields. This is probably why my achievement was so highly regarded.”

The collaborative research group chose Koshihikari, a Japonica type cultivar, and Habataki, an Indica cultivar, as the parent plants for the breeding work. Habataki carries more grains than Koshihikari, and the stem length is shorter. Rice plants are more liable to lodging under wind and heavy rain as the number of grains on each head increases, so shorter stem lengths are favorable. “First, we hybridized Koshihikari and Habataki to create a series of strains with a variety of characteristics, including higher grain numbers, lower grain numbers, shorter stem lengths, and longer stem lengths. Among them, those with higher grain numbers, for example, could be extensively investigated with a special technique known as quantitative trait locus (QTL) analysis. In this way it was possible to find which of the 12 chromosomes of rice carries the gene for the number of grains on each head, and to determine its precise location on that chromosome. Detailed analysis identified this gene as cytokinin oxidase 2 in the Gn1 region in the upper arm of chromosome 1.” In the same way Sakakibara identified the gene for stem length. These analyses were undertaken mainly by Nagoya University and Honda Research Institute Japan Co., Ltd, who are joint research partners.

This was the world’s first identification of the gene for the number of rice grains per head. However, the mechanism behind the involvement of cytokinin oxidase 2 in the grain number remained unknown. Sakakibara clarified it, finding that cytokinin oxidase 2 is a gene for the production of an enzyme that degrades the plant hormone cytokinin.

Cytokinin is responsible for various functions, including promoting cell division, regulating the cell cycle, inhibiting aging, and activating lateral bud growth. “Cytokinin oxidase 2 is expressed in the flower buds and degrades cytokinin. We found that the expression level of cytokinin oxidase 2 was lower in the Habataki flower buds than in those of Koshihikari. As a result, cytokinin escapes degradation, resulting in increased contents of the plant hormone in the flower buds. Hence, cell division is activated, the number of flowers increases, and the number of grains per head increases.”

The mechanism was thus explained. However, the joint research group had one other task: to create rice plants with higher grain numbers and shorter stem lengths.

The result was that plants having the gene regions for a higher number of grains per head and a shorter stem length introduced by hybridization had 20% more grains and a stem length 18% shorter than in conventional Koshihikari plants. These were just as Sakakibara had expected. “Our paper reporting this result greatly pleased those studying cytokinin,” says Sakakibara. “Cytokinin studies have mainly involved basic research, but our achievement proved to be of importance that might lead to agricultural applications, including increased crop yields, which brought a boost in cytokinin research.”

Controlling cytokinin
If cytokinin biosynthesis or activation is artificially adjustable, it might also be possible to improve crop yields easily. Although this is a natural expectation, at that time much remained unknown about the biosynthesis, degradation, activation, and inactivation of cytokinin, even though it had been discovered nearly 50 years ago. Sakakibara was impressed by the necessity for a better understanding of cytokinin. At a meeting of an academic association, he talked with Junko Kyozuka, then Associate Professor at the University of Tokyo’s Graduate School of Agricultural and Life Sciences. She had discovered a rice mutant bearing fewer flowers and had identified the causal gene, but she said that its function remained unclear. Sakakibara says, “Any abnormality in flower bud formation directly affects the yield, so it seemed that she approached me with the suspicion that the abnormality was related to cytokinin. In those days, everyone realized that our team was leading the world in cytokinin analytical technology.”

Examination revealed that the causal gene, named LOG, produced an enzyme that activates cytokinin. “Little had been known about cytokinin activation. Although science textbooks taught that cytokinin becomes activated in two steps, what happens in reality was unknown.”

Another notable finding was that LOG activated cytokinin in one step. The smaller number of flowers is attributed to the insufficient progress of cell division at the growth point, a key factor for flower bud differentiation, because of the absence of cytokinin activation as a result of the mutation in LOG.

“In 2005 we showed that the absence of cytokinin degradation resulting from a mutation in the degradation enzyme increases the yield,” explains Sakakibara. “In 2007 we demonstrated that the absence of cytokinin activation as a result of a mutation in the activation enzyme decreases the yield. The two findings provided evidence that cytokinin is critical in determining rice yields in terms of both degradation and activation.”

Is it therefore possible to control the biosynthesis, activation, and degradation of cytokinin? “Cytokinin is structurally a very simple compound. However, it is regulated in a very complex way in the cells. Although our understanding of its biosynthesis, activation, and degradation is gradually being improved, much remains unknown until we can control cytokinin freely.” Sakakibara is planning to elucidate all aspects of cytokinin using rice, as well as mouse-ear cress. The results are certain to make significant contributions to increasing the yields of other crops, such as wheat and corn.

Using mosses for environmental remediation from contamination heavy with metals
A new joint research program using ‘mosses’ began for the Biodynamics Research Team in April 2008. Initially, the program started as part of the National Project on the Combined Treatment and Recycling of General and Industrial Waste and Biomass (fiscal years 2003–2007), a leading national project sponsored by Japan’s Ministry of Education, Culture, Sports, Science, and Technology. Mosses can survive severe environments, some of which are even capable of growing in places contaminated by heavy metals. These mosses are known to accumulate heavy metals in their cells.

“Among the members of our team is Misao Itouga, a researcher who specializes in mosses. Wandering about with a spoon and capsules in his pockets, he collects any rare mosses he stumbles upon. Once, he collected a sample of Funaria hygrometrica from a waste treatment site and found it to accumulate lead selectively at high concentrations up to 50 to 60 tens of percent (Fig. 3). He hit upon the interesting idea that the moss could be used for environmental remediation. Another moss, Scopelophila cataractae, is known to accumulate copper selectively. However, no one has extensively investigated the potential of mosses for accumulating heavy metals.”

After completion of the national project in March 2008, Sakakibara was looking for a new partner for joint research on mosses, and he came across DOWA Holdings Co., Ltd, a company engaged in the mining and ore refining business, which showed interest in his project.

There are two major themes for the joint research. One is to discover other mosses that accumulate heavy metals at high concentrations. “We visit everywhere we can to collect a wide variety of mosses, and examine them for their potential to accumulate heavy metals.” The team has also begun to study the induction of mutations by means of heavy-ion beams from the RIKEN ring cyclotron and to look for mutants that accumulate any heavy metal that is not currently known to accumulate in normal individuals. “If a mutant capable of accumulating a precious metal, also known as a rare metal, is discovered, it will find commercial applications and be in high demand.” The mechanism by which mosses selectively accumulate metals is of course also interesting. “If the mechanism is explained, it will become possible to artificially accumulate any heavy metal desired.”

The other theme is to develop a purifier for waste water contaminated with heavy metals. The developmental project is targeted for completion in three years. The apparatus comprises a vessel containing protonemata, the earliest, thread-like, stage of growth of moss plants of Funaria hygrometrica. When contaminated water is fed in from above, purified water comes out from the bottom.

Moss research is quite different from cytokinin research. “Almost nothing is known about mosses, but repeated shots at the target could lead to a big hit,” says Sakakibara with a laugh. It is important to him that any research theme be scientifically fascinating. If this were not so, researchers would never find the motivation to do the research. He is very happy in his current circumstances, which allow him to conduct research with actual applications in mind, based on basic research. He is keen to continue investigations that will actually help to resolve the coming food crisis and environmental issues.

About the researcher
Hitoshi Sakakibara was born in Aichi, Japan, in 1965. He was graduated from the School of Agricultural Sciences, Nagoya University in 1988. He obtained his PhD in agriculture from Nagoya University in 1995. He was appointed as Research Associate (1992) and then Assistant Professor (1995) of the School of Agricultural Sciences, Nagoya University. He joined the Plant Science Center in October 2000 as Head of the Laboratory for Communication Mechanisms. He continues his research project in the second phase of the Plant Science Center as Team Leader of the Biodynamics Research Team and as Group Director of the Plant Productivity Systems Research Group. Since graduate school he has been conducting biochemical and molecular physiological studies on the regulation and mechanism of expression of nitrogen assimilatory genes, cytokinin-mediated nitrogen signaling, and cytokinin biosynthesis.
Hitoshi Sakakibara
Group Director
Plant Productivity Systems Research Group
Plant Science Center