Prof. Yoshikatsu Matsubayashi, Dr. Ryo Tabata, Ms. Kumiko Sumida, and Assistant Prof. Hidefumi Shinohara at the Division of Biological Science, Graduate School of Science, Nagoya University, have discovered a novel plant peptide hormone "CEP." The peptide hormone is produced by plant roots in response to a nitrogen (N)-starved condition on one side of plant root and results in increased nitrate uptake on the other side of the root. This action suggests that plants can skillfully adapt to fluctuating N-levels in their environment. Applications to N-starvation and crop production are also expected.
This research was published in Science on October 17, 2014.→ Nagoya University Press Release

Plant hormones play an essential role in plant growth.

Plant hormones are molecules that are produced by plants themselves and possess the ability to modulate physiological activity and signaling. Organic compounds may be known as the general form of plant hormones, although recently peptide hormones (peptide molecules) that influence cell-to-cell communication have also been identified.

―Discovering unknown hormones and revealing their roles in plant growth; it is an intriguing field of study.

In general, researchers may have their own curiosity about plant physiological phenomena, such as "why flowers bloom," and the mechanism would be revealed on the research process, in most cases by chance.

"We have expertise in the field of molecules."

Prof. Yoshikatsu Matsubayashi et al. at the Graduate School of Science, Nagoya University, have explored the key points of plant growth in terms of substances or molecules. It can be initially complicated to understand what type of functions various substances have; therefore, the research group targets signaling molecules and proceeds by using various clues from various directions.

One of the clues is to analyze signaling molecular groups going in and out of the apoplast between the cells. The research group has established its own technologies for high precision analyses, which provides the strength for advancing their research.

In addition, the research group has produced the cell lines expressing the receptor candidates for hormones, preparing for several years in the laboratory. A library of these cells has been built and stored in the freezer at -80 °C for use at any time. Till date, approximately 200 lines have been produced, which appears to account for the majority of the possible receptor candidates.

"We aim to reveal each plant physiological phenomenon at the molecular level by identifying ligand-receptor pairs that relate to plant morphogenesis and environmental adaptation."

Not focusing on a particular plant physiological phenomenon to identify the responsible genes, Prof. Matsubayashi and his colleagues address particular ligand-receptor pairs that relate to the physiological phenomenon. "Changing the viewpoint" leads to breakthroughs in discovering interesting phenomena and mechanisms that have been overlooked so far.

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Amino acids combine in numerous ways to form various types of peptides, with some acting as ligands, such as the so-called "peptide hormones." Even if we limit the peptide length to 50-150 amino acids, there are approximately 1,000 secreted peptides in the genomes of plants.

"Most organisms have several spare copies of a gene so as not to lose the gene function."

The copies of a gene show the same feature of the function despite having slightly different sequences. In the laboratory, comparing a hormone-gene-deficient strain with a wild type, the hormonal function may be explained as a phenomenon. However, usually no differences occur because the spare genes compensate for the target peptide gene's function. Herein, we have the difficulty of peptide hormone research.

"We have been targeting peptide hormones that have been overlooked."

If the peptide hormones have a number of gene copies, those genes are possibly more important. In 2010, Prof. Matsubayashi et al. focused on the enzymes that create special structures of the peptide molecules (post-translational modification enzymes), and then overcame the challenge. Because such modifications are critical for the functions of individual peptide hormones, phenotypic analysis of loss-of-function mutants of these posttranslational modification enzymes allows the presence of novel peptide hormones to be uncovered. They discovered a hormone necessary for root growth, root meristem growth factor (RGF) (Matsuzaki et al. Science (2010) 329: 1065-7).

In this study also, a focus on the molecular structure was critical to the discovery.

From the genetic information deciphered from the Arabidopsis genome, Prof. Matsubayashi and his research group singled out a peptide group candidate (Figure 1). Because the candidate had almost the same amino-acid sequence in the C-terminus, it was named C-terminally encoded peptide (CEP) (Ohyama et al. Plant J. (2008) 55: 152-60).

Figure 1. Amino-acid sequence in the C-terminus, or CEP. Black and gray colors show the highest and higher matches in the allignment, respectively. (Figure: by courtesy of Prof. Matsubayashi)

"Many of the CEPs were found specifically expressed in the roots, although initially we did not know their role."

Dr. Ryo Tabata, from the same research group, started analyzing the function of CEPs as a peptide hormone candidate. However, because of the large number of copies of CEPs that were present, it was difficult to make all of the CEP-deficient plants.

"I began to examine the receptor side."

Dr. Tabata proceeded the round-robin of binding experiments between CEPs and receptor candidates from the receptor library established in the laboratory. Fortunately, after examining approximately 40 candidates, two CEP receptors (CEPRs) were found.

Subsequently, the prepared CEPR-deficient strains were compared with wild type strains. In the CEPR-deficient strain, morphological changes such as lateral root extension, yellowing leaves, and dwarfing were observed (Figure 2). These changes were often observed when nitrogen (N) was deficient in the plants; however, it was necessary to examine whether CEPs were involved in any aspect of the N-response.

Figure 2. Comparison between wild type strains and CEPR-deficient strains grown on the N-rich medium. (Figure: by courtesy of Prof. Matsubayashi)

"I looked through a lot of papers to find any other description of the phenomenon."

After several hypotheses and repeated experiments to verify the CEP function, finally it was revealed that the role of the CEP-CEPR pair was related to "systemic N-demand signaling."(Gloria M. Coruzzi et al. PNAS (2011) 108: 18524-9).

Systemic N-demand signaling refers to a mechanism for capturing extra N-nutrition on one side of the plant root when the other side has N-shortage. This ensures that even if the distribution of nitrate is uneven in the soil, the action of CEP-CEPR enables an individual plant to maintain optimal the uptake of nitrate ions.

The mechanism and function of CEP are summarized (Figure 3):

If some roots recognize N-starvation, the expression level of CEP family peptides in the roots increases, the CEP peptides are passed through the xylem and is then recognized by the receptor CEPR expressed in the veins of leaves.

Furthermore, the secondary signal transfers to other roots within the plant, which initiate the uptake of extra N to compensate for the local N-deficiency.

CEP is the peptide hormone used to perceive and compensate for "hunger," and the growth of and individual plant is maintained by the role of CEP.

Figure 3. The mechanism and function of CEP. (Figure: by courtesy of Prof. Matsubayashi)

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"It is better not to have the same approach as everyone else so that you may discover something that has been overlooked."

This great discovery tells us the importance of various ways of thinking.

The result also convinces us of the ingenuity and cleverness of plants for environmental adaptation, which starts a new concept of plant growth by chemical control. The current environmental issues and problems of food shortage, which are strongly related to our life, could be addressed in principle.

Although plants live quietly and daintily,

―dynamic signaling in plants and their wise growth continue to fascinate us.

(Ayako Umemura)

Researchers featured in this article

Dr. Yoshikatsu Matsubayashi【Professor, Graduate School of Science, Nagoya University】

Dr. Yoshikatsu Matsubayashi was born in 1971 in Mie prefecture. He completed a doctorate course at the Graduate School of Bioagricultural Sciences, Nagoya University, in 1997. He worked as an Assistant Professor and Associate Professor at Nagoya University and then moved to the National Institute for Basic Biology as a Professor in 2011. He started at the present post since April 2014.

Dr. Matsubayashi has received a number of awards during his career:

JSBBA Award for Young Scientists (2001);
Japanese Society of Plant Physiologists Young Investigator Award (2008);
Molecular Biology Society of Japan Mitsubishi Chemical Award (2010).

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Dr. Matsubayashi pursues photography as a hobby. His room is adorned with photographs of auroras taken in northern Sweden, and I was attracted to their mysterious colors.
Dr. Matsubayashi says that concentration achieved by switching on and off is necessary in research so as not to miss any opportunistic situation. The aurora photographs exemplify this opportunistic concept as he was fortunate to take them at the best time over the last half year. Luck is also a part of ability. I expect he will keep leading his research group and making further achievements in future work (by AU)

Dr. Ryo Tabata【Postdoctoral researcher, Graduate School of Science, Nagoya University】

Dr. Ryo Tabata completed a doctorate course at the Graduate School of Bioagricultural Sciences, Nagoya University, in 2007, and then started working as a COE researcher at the same school. In 2010, Dr. Tabata moved to the Graduate School of Science at The University of Tokyo as a project researcher and also to the Graduate School of Natural Sciences at Kumamoto University as a fellow of The Ministry of Education, Culture, Sports, Science and Technology. In 2013, he started working at the National Institute for Basic Biology as a research fellow. Since 2014, he has worked at his current post at Nagoya University.

Dr. Tabata received an Award for Excellence to Authors Publishing in Bioscience, Biotechnology, and Biochemistry in 2008.

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Dr. Tabata's hobby is fishing. "As long as there are many friends and colleagues, who like fishing, around me, I will continue to enjoy it" he says. I think he is lucky because of his virtue.
Dr. Tabata likes to think about where the fish are and to imagine them in the sea. He added "it's important to think the invisible things, and I like that." I may find out why he likes both fishing and research. I am looking forward to his further achievements in his future research (by AU)

Links

• Laboratory HP　http://www.bio.nagoya-u.ac.jp/~b2/index.html

• Link to this article (from a search-result)
  http://www.sciencemag.org/content/346/6207/343.abstract

Ryo Tabata, Kumiko Sumida, Tomoaki Yoshii, Kentaro Ohyama, Hidefumi Shinohara, and Yoshikatsu Matsubayashi
Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling.
Science 346: 343 (2014).
（First published on 17 October 2014; DOI: 10.1126/science.1257800）

• Link to related article1 (from a search-result)
  

  http://www.sciencemag.org/content/329/5995/1065.abstract

Yo Matsuzaki, Mari Ogawa-Ohnishi, Ayaka Mori and Yoshikatsu Matsubayashi

Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis.

Science 329: 1065 (2010).

（First published on August 27, 2010; DOI: 10.1126/science.1191132）

• Link to related article2 (from a search-result)
  http://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2008.03464.x/abstract

Kentaro Ohyama, Mari Ogawa and Yoshikatsu Matsubayashi

Identification of a biologically active, small, secreted peptide in Arabidopsis by in silico gene screening, followed by LC-MS-based structure analysis.

Plant J. 55: 152 (2008).

(Published on June 28, 2008; DOI: 10.1111/j.1365-313X.2008.03464.x)

• Link to related article3 (from a search-result)
  http://www.pnas.org/gca?gca=pnas%3B108%2F45%2F18524&submit=Get+All+Checked+Abstracts

Sandrine Ruffel, Gabriel Krouk, Daniela Ristova, Dennis Shasha, Kenneth D. Birnbaum,　and Gloria M. Coruzzi
Nitrogen economics of root foraging: Transitive closure of the nitrate-cytokinin relay and distinct systemic signaling for N supply vs. demand.
PNAS. 108: 18524 (2011).
(Published on October 24, 2011; doi:10.1073/pnas.1108684108)