Masaru Ohme-Takagi (Invited Senior Researcher and Professor at Graduate School of Science and Engineering, Saitama University), Yukari Nagatoshi (former AIST Postdoctoral Researcher, currently Researcher at the Japan International Research Center for Agricultural Sciences) and others, the Plant Gene Regulation Research Group, the Bioproduction Research Institute (Director: Tomohiro Tamura), the National Institute of Advanced Industrial Science and Technology (AIST; President: Ryoji Chubachi), have succeeded in enhancing ozone resistance of plants by adjusting the opening of stomata on plant leaves, in collaboration with the Japan International Research Center for Agricultural Sciences (JIRCAS; President: Masa Iwanaga), Saitama University (President: Hiroki Yamaguchi), the National Institute for Environmental Studies, Nagoya University, Okayama University, and RIKEN.

In recent years, atmospheric pollution has caused extensive damage to agricultural crops and forests. The researchers discovered that expressing chimeric repressors of the transcription factors that control the development of chloroplasts in plants (transcription factors GLK1 and GLK2) significantly improves resistance to ozone, which is an atmospheric pollutant. In plants which were made to express the chimeric repressors of these transcription factors, the researchers found that stomata close slightly, and the transcription factors GLK1 and GLK2 affect factors relating to the stomatal movements. It is expected that if stomatal aperture can be properly adjusted using these transcription factors, it would contribute to the development of crops that are tolerant of environmental stress, such as atmospheric pollution and drought.

Details of the results will be published in an American scientific journal, Proceedings of the National Academy of Sciences, on March 28, 2016 (Eastern Standard Time).

GLK1SRDX transformed Arabidopsis thaliana showed tolerance to high concentrations of ozone (left) and GLK1OX transformed Arabidopsis thaliana showed hypersensitive to high concentrations of ozone (right).

Atmospheric pollution is advancing on a global scale. Near-surface ozone is an air pollutant, and a major component of photochemical smog. High concentrations of ozone not only damage our health, but also inflict enormous damage on plants, causing the decline of forests and decreased agricultural crop yield. It has been predicted that ozone concentration will continue to increase, so there are expectations that technologies for understanding of the response mechanism of plants to ozone and improvement of ozone resistance in agricultural crops will be developed.

AIST and Saitama University have conducted research on the transcription factors of plants in order to effectively use the original functions held by plants, with the goal of solving environmental problems and foods problems. So far, using the AIST original gene silencing technology, “CRES-T Method”, the two organizations have achieved results in functional analysis and usable technology development of transcription factors in model plants and commercial plants. The research has also demonstrated that the CRES-T method is useful for granting stress-resistance and modifying the form of commercial plants, such as producing salt-tolerant rice and polypetalous cyclamen. JIRCAS has achieved outstanding results in the analysis of signaling pathways of environmental stress response and the development of tolerant crops.

Both parties used the CRES-T method to identify new transcription factors that improve ozone-resistance of plants and to analyze their mechanisms.

When high concentration ozone is absorbed by a plant, it causes the failure of leaves. This failure of leaves due to ozone has an impact on deterioration in the quality of leafy vegetables, as well as inhibition of growth and loss of yield associated with decreased photosynthetic capacity, making it a serious type of ozone damage. Therefore, the researchers attempted to search for transcription factors that improve ozone resistance (reduce the failure of leaves). Using the CRES-T method, they prepared transformants of Arabidopsis thaliana expressing chimeric repressors that inhibited the function of approximately 1,500 transcription factors. Approximately 30,000 individual transformed Arabidopsis thaliana were exposed to high concentration ozone and the researchers selected the ozone tolerant plants. From these plants, they identified transcription factors GLK1 and GLK2 which significantly affect ozone tolerance. Arabidopsis thaliana expressing a chimeric repressor of GLK1 or GLK2 (GLK1SRDX or GLK2SRDX) did not show failure of leaves due to ozone even when exposed to high concentration ozone (0.3 ppm) for 7 hours, indicating ozone tolerance. Additionally, it was found that the surface temperature of leaves was high (that is, less transpiration) and the stomata were slightly closed in these transformants of Arabidopsis thaliana (Fig. 1). Stomata are small pores in the surface of plant leaves, and play roles in the uptake of carbon dioxide necessary for photosynthesis and water regulation in the body of the plant. Stomata are also involved in the uptake of air pollutants that are harmful to plants. In Arabidopsis thaliana expressing the chimeric repressor of GLK1 or GLK2 (GLK1SRDX or GLK2SRDX), it is thought that the transformants indicated ozone tolerance by the stomata slightly closing, reducing ozone uptake. Conversely, in Arabidopsis thaliana with enhanced GLK1 and GLK2 functions (GLK1OX and GLK2OX), it was found that the stomata were slightly more open, making them weaker to ozone. These indicate that it is possible to increase the yield of crops by properly adjusting the stomatal aperture, so GLK1 and GLK2 are expected to be useful.

Thus far, it has been known that GLK1 and GLK2 are involved in the development of chloroplasts, but their involvement in the stomatal movements was not known. From detailed analysis, the researchers found that in Arabidopsis thaliana expressing the chimeric repressor of GLK1 (GLK1SRDX), gene expression and activity of inwardly rectifying potassium channels which are a necessary factor for opening stomata were reduced. In addition, ozone tolerance was improved in Arabidopsis thaliana expressing the GLK1 chimeric repressor (GLK1SRDX) only in cells which adjust the stomatal aperture (guard cells). From these facts, it was found that the transcription factors GLK1 and GLK2 also affect the expression of genes relating to the stomatal movements. The CRES-T method is useful for a variety of commercial plants as well, so there is a possibility to grant air pollutant resistance to crops using these technologies.

Figure 1: GLK1SRDX transformed Arabidopsis thaliana, with high leaf surface temperature and low transpiration rate

While stomata are involved in the uptake of air pollutants and water loss from the body of plants, they are also involved in photosynthesis activity and growth. In the future, the researchers aim for technology leading to the development of more productive plants by controlling and expressing the GLK1 chimeric repressor (GLK1SRDX) in guard cells only when a plant is subjected to stress such as ozone or drought, to properly adjust the stomatal aperture.