Through photosynthesis, they use sunlight and carbon dioxide to make food, belching out the oxygen that we breathe as a byproduct. This evolutionary innovation is so central to plant identity that nearly all land plants use the same pores — called stomata — to take in carbon dioxide and release oxygen.

Stomata are tiny, microscopic and critical for photosynthesis. Thousands of them dot on the surface of the plants. Understanding how stomata form is critical basic information toward understanding how plants grow and produce the biomass upon which we thrive.

In published May 7 in the journal , a 91̽��-led team describes the delicate cellular symphony that produces tiny, functional stomata. The scientists discovered that a gene in plants known as MUTE orchestrates stomatal development. MUTE directs the activity of other genes that tell cells when to divide and not to divide — much like how a conductor tells musicians when to play and when to stay silent.

“The MUTE gene acts as a master regulator of stomatal development,” said senior author , a 91̽��professor of biology and investigator at the . “MUTE exerts precision control over the proper formation of stomata by initiating a single round of cell division — just one — in the precursor cell that stomata develop from.”

Stomata resemble doughnuts — a circular pore with a hole in the middle for gas to enter or leave the plant. The pore consists of two cells — each known as a guard cell. They can swell or shrink to open or close the pore, which is critical for regulating gas exchange for photosynthesis, as well as moisture levels in tissues.

“If plants cannot make stomata, they are not viable — they cannot ‘breathe,’” said Torii, who also is a professor at Nagoya University in Japan.

Torii and her team investigated which genes governed stomata formation in Arabidopsis thaliana, a small weed that is one of the most widely studied plants on the planet. Past research by Torii’s team and other researchers had indicated that, in Arabidopsis, MUTE plays a central role in the formation of stomata. The MUTE gene encodes instructions for a cellular protein that can control the “on” or “off” state of other plant genes.

The researchers created a strain of Arabidopsis that can artificially produce a lot of the MUTE protein, so they could easily identify which genes the MUTE protein turned on or off. They discovered that many of the activated genes control cell division — a process that is critical for stomatal development.

In Arabidopsis, as in nearly all plants, stomata form from precursor cells known as guard mother cells, or GMCs. To form a working stoma — singular for stomata — a GMC divides once to yield to paired guard cells. Since their data showed that MUTE proteins switched on genes that regulated cell division, Torii and her team wondered if MUTE is the gene that activates this single round of cell division. If so, it would have to be a tightly regulated process. The genetic program would have to switch on cell division in the GMC, and then quickly switch it right back off to ensure that only a single round of division occurs.

Torii’s team showed that one of the genes activated by the MUTE protein to its DNA is CYCD5;1, a gene that causes the GMC to divide. The researchers also found that MUTE proteins turn on two genes called FAMA and FOUR LIPS. This was an important discovery because, while CYCD5;1 turns on cell division of the GMC, FAMA and FOUR LIPS turn off — or repress — the cell division program.

“Our experiments showed that MUTE was turning on both activators of cell division and repressors of cell division, which seemed counterintuitive — why would it do both?” said Torii. “That made us very interested in understanding the temporal regulation of these genes in the GMC and the stomata.”

Through precise experiments, they gathered data on the timing MUTE activation of these cell division activators and repressors. They incorporated this information into a mathematical model, which simulated how MUTE acts to both activate and repress cell division in the GMC. First, MUTE turns on the activator CYCD5;1 — which triggers one round of cell division. Then, FAMA and FOUR LIPS act to prevent further cell division, yielding one functional stomata consisting of two guard cells.

“Like a conductor at the podium, MUTE appears to signal its target genes — each of which has specific, and even opposite, parts to play in the ensuing piece,” said Torii. “The result is a tightly coupled sequence of activation and repression that gives rise to one of the most ancient structures on land plants.”

Co-lead authors on the paper are Soon-Ki Han, a former 91̽��postdoctoral researcher now at Nagoya University, and 91̽��postdoctoral researcher Xingyun Qi. Additional co-authors are Jonathan Dang, Kristen Miller and Eun Deok Kim at the UW; Kei Sugihara and Takashi Miura at Kyushu University; and Takaho Endo at the RIKEN Center for Integrative Medical Sciences. The research was funded by the National Science Foundation, the Gordon and Betty Moore Foundation and the HHMI.

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For more information, contact Torii at 206-221-5701 or ktorii@uw.edu.

Grant numbers: MCB-0855659, GBMF-3035.

A lab-designed hormone could unlock mysteries harbored by plants.

By developing a synthetic version of the plant hormone auxin and an engineered receptor to recognize it, 91̽�� biology professor and her colleagues are poised to uncover plants’ inner workings.

The new work, described in a published online Jan. 22 in , is a “transformative tool to understand plant growth and development,” said Torii, who is also an with the . That understanding may have big agricultural implications, raising the possibility, for instance, of a new way to ripen fruits such as strawberries and tomatoes.

To plants, the hormone is king. Among many other jobs, auxin helps sunflowers track sunlight, roots grow downward and fruits ripen. This wide range of jobs, as well as the fact that every cell in a plant can both produce and detect auxin, makes it tricky to tease apart the hormone’s various roles.

“It’s been a huge mystery as to how such a simple molecule can do so many different things,” said Torii, who is senior author on the paper.

She and her colleagues set out to create a new way to study plants’ responses to auxin by designing a lab-made version of the hormone that can be precisely controlled. Working with , the researchers added a little bump to auxin’s structure — hydrocarbon rings that auxin doesn’t normally contain. The researchers then tweaked plants’ auxin receptor, a protein that sits on the outside of plant cells and detects auxin. They removed a bulky amino acid from the receptor, creating a perfect-sized hole that cradles the lab-made auxin.

That simple switch, called a “bump and hole” strategy, “is really elegant, actually,” said Torii.

Next, the researchers tested whether this matched set — the synthetic auxin and the synthetic receptor — could do the same jobs as the cells’ natural auxin/receptor pair. The intricately designed system worked beautifully, based on results from experiments on roots.

Normally, roots exposed to auxin stop growing down, and instead grow sideways by activating stem cells that break out of the main root. The process, called lateral root development, is comparable to aliens bursting through stomachs, said Torii. After detecting synthetic auxin, Arabidopsis plants genetically engineered to produce the synthetic auxin receptor behaved just like normal — growing the same sideways baubles of root branches.

What’s more, roots that didn’t have the synthetic auxin receptor were essentially “blind” to synthetic auxin, proof that the artificial hormone is detected by only the artificial receptor. Torii and her colleagues call this switch to synthetic auxin “chemical hijacking” — a well-controlled takeover that now will allow researchers to tease apart the tangled web of auxin’s jobs in plants.

With their system up and running, the researchers tested a long-standing question in plant biology. Scientists knew that germinating seedlings use auxin to grow quickly. But the identity of the exact receptor that allows this process to happen wasn’t settled.

The scientific community had a suspect in mind. Torii’s team produced a plant that lacked an auxin receptor called TIR1, and instead possessed a synthetic version. When exposed to artificial auxin, these seedlings began to grow rapidly, behaving exactly as if they had the normal receptor. The results suggest that seed elongation does indeed happen through the TIR1 receptor.

Other fundamental scientific questions can be addressed with this system, Torii said, such as auxin’s role in corn ripening and in opening the stomata, the structures that let plants breathe.

One day, synthetic auxin might even find a place in agriculture. Auxin is currently sprayed on fruits to hasten ripening. But in high concentrations, the hormone can act as a plant-killing herbicide. Fruits engineered to carry the synthetic receptor could be ripened with the synthetic auxin hormone, Torii said, eliminating the need to spray auxin indiscriminately. But, she cautions, much more testing needs to be done before a synthetic hormone system can be used for growing food.

Naoyuki Uchida and Koji Takahashi at Nagoya University are co-lead authors on the paper. Co-authors on the study are Rie Iwasaki, Ryotaro Yamada, Masahiko Yoshimura, Hua Zhang, Mika Nomoto, Yasuomi Tada, Toshinori Kinoshita, Kenichiro Itami and Shinya Hagihara at Nagoya University; Takaho Endo with the ; and Seisuke Kimura at . The research was funded by the Japan Society for the Promotion of Science; the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Japan Science and Technology Agency; the Howard Hughes Medical Institute; and the .

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For more information contact Torii at ktorii@uw.edu.

Adapted from a by the Howard Hughes Medical Institute.

Grant numbers: JP26291057, JP16H01237, JP17H06476, JP16H01462, JP17H03695, JP17KT0017, JP26440140, JP15H05956, JP17H06350, JP16H01472, S1511023, JPMJPR15Q9, JPMJER1302, GBMF3035.

As they published online June 17 in , the team led by 91̽��biology professor and senior author made its discovery as they explored how plants organize cellular structures on their surface.

Like other multicellular creatures, plants must coordinate activity among many different types of cells and tissues. Messages, demands, warnings and alerts shuttle among cells near and far. These messages determine what jobs cells take on and how they work together to build and maintain tissues and organs. As plants grow, they also use this information to decide where new structures like leaves or roots should go.

Torii, lead author Jin Suk Lee and their colleagues focused on how plants decide where to place stomata: tiny, two-cell openings on the surface that connect the plant’s interior with the outside world. Critical for water and gas exchange, stomata develop on the plant’s surface based largely on signals they receive from neighboring cells.

“Stomata are so important for plant productivity,” said Torii, who is also an with and . “They’re small but have a big impact.”

Plants must grow and distribute their stomata evenly on the surface because too many or too few can disrupt water balance or photosynthesis.

Lee and Torii studied two signals that plant cells release to control where stomata go. These signals are actually proteins, or small molecules that help cells do work and communicate with one another. One is called Stomagen, which promotes stomata development. The other protein messenger — known by its acronym EPF2 — opposes Stomagen by preventing stomata formation.

“My lab had previously identified many key factors that control stomata development and enforce proper stomata patterning,” said Torii. “We had described EPF2 and another group identified Stomagen. But we did not know how plant cells balanced these two signals.”

Her team wanted to understand how cells near the plant’s surface recognize Stomagen and EPF2 and how they decide which signal to obey. They studied these questions in a small mustard weed called Arabidopsis thaliana, which researchers including Torii have long studied to understand how plant cells organize themselves into a functional body.

The researchers studied how messages from Stomagen and EPF2 are transmitted in mutant Arabidopsis plants that lacked certain cell surface proteins. Like a person standing in the doorway of a house, these surface proteins have one foot planted outside and the other inside and are important conduits for conveying information and messages into the cell.

They discovered that Stomagen and EPF2 actually compete for access to the same surface proteins that can transmit either a stomata-promoting or stomata-repressing message into the cell based on which signal molecule binds to them. If Stomagen attaches, then the cell receives a message to create a stomata. If EPF2 attaches instead, the surface proteins tell the cell to shut down stomata development.

“The cell has these competing signals that it has to interpret, and it uses the same surface protein for both,” said Torii. This type of signal delivery system — where two opposing messages compete directly for access to the same proteins — exists in animals. But this type of antagonistic signaling has never been seen in plants, she added.

This is a particularly surprising finding because Stomagen and EPF2 look very similar to one another. They differ in only a few key qualities. Yet those small differences amount to big differences in the messages they deliver to cells.

The discovery sheds light on the mechanisms that cells employ to detect and process messages — including conflicting signals — from the outside world. In the future, Torii would like to understand how the pro-stomata and anti-stomata messages act once they’re inside plant cells.

“This paper is just the beginning,” she said.

Lee, who conducted this research as a postdoctoral fellow in Torii’s lab, is now in Montreal. Other co-authors are Marketa Hnilova with the UW’s and researchers Michal Maes, Ya-Chen Lisa Lin, Aarthi Putarjunan, Soon-Ki Han and Julian Avila.

This research was funded by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (GBMF3035).

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For more information, contact Torii at 206-221-5756 or ktorii@uw. edu.