College of Life Science Demonstrates Strong Research Capacity

Recently, the faculty and students of the NTU College of Life Science have been demonstrating a strong capacity for research. Their studies have shed light on the future improvement of wood processing; the mechanism of a protein complex and its relation to enzyme catalysis and molecular regulation in photomorphogenesis; the effect of the interaction among genomes, genetic mechanisms, and environmental adaptation on organismal evolution; the mechanism of homologous recombination-mediated DNA repair and its potential in cancer prevention and treatment; the mechanistic role of a protein in homologous recombination-mediated DNA double-strand break repair; and the bird pollination system in East Asia.

In 2017, Associate Prof. Hung-Yuan (Peter) Chi (冀宏源) from the Institute of Biochemical Sciences received the Junior Research Investigators Award from Academia Sinica, and two undergraduate students Chia-Yi Lee (李佳怡) and Jing-Yi Lu (盧璟誼) from the Department of Life Science won the College Student Research Creativity Award from the Ministry of Science and Technology (MOST) under the guidance of their advisors.

Below are the more detailed introductions of the six recent outstanding studies featured in this article:

The Real Master Regulators above the Master Regulators: Homeostasis of the Wood Formation Regulation

By Assistant Prof. Ying-Chung Lin (林盈仲) from the Department of Life Science

Wood is a renewable and abundant raw material for pulping, energy, and solid wood products. During industrial manufacturing, some of the components in wood decrease production efficiency or product quality. For example, short wood fibers could lead to low paper quality, and lignin in wood causes low efficiency in the conversion of wood to bioethanol. In the past decades, reengineering wood composition to obtain a better raw material for industrial production has become a worldwide issue. Wood formation involves a complex development of many different cell types, which includes numerous regulations. However, little is known about the regulation of wood formation leading to the difficulties and barriers for wood composition engineering. In this study, we report key regulators involved in wood formation, and provide novel knowledge for future improvement of wood quality.

Vascular-related NAC-domain (VND) and secondary wall-associated NAC domain (SND) families have been found as master regulators of secondary cell walls during wood formation. Both families activate downstream transcription factors to create an activation cascade to the wood forming genes. However, this strong activation would stunt plant growth in the absence of a negative feedback regulation. In this study, we used the model woody plant, Populus trichocarpa, and discovered splice variants, SND1-A2^IR and VND6-C1^IR, respectively from the SND1 and VND6 families that can exert as negative regulators (dominant negative) for the two families. SND1-A2^IR and VND6-C1^IR lack DNA binding and transactivation domains, but retain protein-protein interaction domains. By forming non-functional heterodimers with SND1 and VND6 family members, SND1-A2^IR and VND6-C1^IR suppressed the gene expression of SND1 and VND6, as well as their activation of downstream gene MYB021. SND1 and VND6 are found to be expressed in fiber and vessel cells (Figure 1), respectively, in non-woody plant xylem tissue without any splice variants. In P. trichocarpa, both SND1 and VND6 are expressed in fiber and vessel cells. Furthermore, SND1-A2^IR and VND6-C1^IR cross-regulate both the SND1 and VND6 families (Figure 2). Such a splice variant-regulating mechanism has not been reported in non-woody plants, suggesting a possible major difference between the secondary cell wall formation mechanisms of woody and non-woody plants.

*Link of the paper: http://www.pnas.org/content/early/2017/10/19/1714422114.full.

Plant Hormone Jasmonate-Isoleucine (JA-Ile) Biosynthesis May be Regulated by Far-Red Light Signaling Based on the FIN219-FIP1 Protein Complex

By Associate Prof. Yi-Sheng Cheng (鄭貽生) from the Department of Life Science, Institute of Plant Biology, and Genome and Systems Biology Degree Program

Light influences plant growth and development. Far-red light and red light can synergistically affect seed germination and photomorphogenesis through the phytochrome photoreceptors in plant cells. A previous study demonstrated that a jasmonate synthetase, FIN219 (FAR-RED INSENSITIVE 219), also known as JAR1 (JASMONATE RESISTANT 1), functions to conjugate jasmonate (JA) to amino acids such as Isoleucine (Ile) in the JA signaling pathway. JA-Ile is an active phytohormone required for plant defense, growth, and development. Therefore, far-red light-coupled JA signaling is controlled by FIN219 activity. In Arabidopsis, FIN219 interacts with the protein FIP1 (FIN219-INTERACTING PROTEIN 1) in the far-red light signaling pathway. However, the mechanism and function of this interaction are unknown. Here, we use several methods, such as X-ray crystallography and biophysical and biochemical analyses, to elucidate the molecular characteristics of the FIN219-FIP1 complex.

From the crystal structure of the FIN219-FIP1 complex, we observed that the orientation of the FIN219 C-terminal domain shifted after binding FIP1 and moving near the active site of FIN219; this domain could then stabilize the binding of substrates, such as JA, Ile, and ATP. Based on this important structural information, we further resolved 12 FIN219-FIP1 structures with different combinations of substrates such as ATP, JA, and the amino acids Ile, Leu, Met, and Val. The structures of FIN219-FIP1 complexes bound to these substrates reveal the preference of FIN219 for Ile over other amino acids. This amino acid preference results from differences in ATP orientation in the substrate binding pocket of FIN219. Furthermore, the binding order of the substrates JA, ATP, and Ile was examined using a quartz crystal microbalance (QCM), a biophysical assay machine. This assay showed that FIN219 first binds to JA and then binds to ATP or Ile. The purpose of the interaction between FIN219 and FIP1 was determined by enzyme kinetics and QCM. Compared with FIN219 alone, the FIN219-FIP1 complex showed 2.3-fold increased K_cat and 2-fold increased V_max, demonstrating that FIN219 enzyme activity was enhanced by FIP1 binding. Finally, we propose a model to explain the changes in conformation and enzyme activity of FIN219 after FIP1 binding. In summary, the detailed mechanism of the interaction of FIN219 with FIP1 provides valuable insights into enzyme catalysis in the GH3 (glycoside hydrolase 3) family and molecular regulation in photomorphogenesis (Figure 3).

Reference:

Chun-Yen Chen, Sih-Syun Ho, Tzu-Yen Kuo, Hsu-Liang Hsieh, and Yi-Sheng Cheng*, (2017). Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation. Proc. Natl. Acad. Sci. U.S.A. 2017 114:E1815-E1824, doi: 10.1073/pnas.1609980114.

*This essay was originally featured in Issue 3 (2017) of Landscape: NTU Research and Development.

The Effect of the Interaction among Genomes, Genetic Mechanisms, and Environmental Adaptation on Organismal Evolution

By Assistant Prof. Cheng-Ruei Lee (李承叡) from the Institute of Ecology and Evolutionary Biology

In an article published in Nature Communications in 2017, Dr. Lee’s team performed big-data analysis on publicly available Arabidopsis thaliana genomes to infer the population history of this species. The results suggest high similarity between the population history of A. thaliana and that of humankind. Anatomically, modern humans expanded from Africa into Eurasia about one hundred thousand years ago, excluding while hybridizing with other human races, namely Neanderthals in Europe and Denisovans in Asia, along the way. The hybridization has major impacts on modern humans’ environmental adaptation. For example, modern Tibetans have inherited a piece of DNA from Denisovans, which helps them adapt to the harsh, high-elevation environments. There are several highly diverged populations within A. thaliana, including a weedy population across Eurasia and several natural populations distributed around native forests in southern Europe. Dr. Lee’s team found that, after the last ice age, the present-day natural populations had expanded from southern to northern Europe following glacier retreat. About ten thousand years ago, one population (the present-day weedy population) became dominant, expanding and excluding other natural populations in Eurasia. During the expansion, the weedy population hybridized with different local populations, creating an interesting pattern we observe today. Individuals near the edge of the species range (i.e., northern Africa, southern and northern Europe, and northern Russia) retain more genetic elements inherited from local natural populations, whereas individuals in mid-latitude Europe are the direct descendants of the weedy population with high genetic similarity. In the Iberian Peninsula, the weedy population has hybridized with and inherited from the natural population a genetic element conferring local adaptation to the specific environments in Iberia. This hybridization therefore allows those weeds, which invaded Iberia from western Europe, to survive the novel environment. This story is therefore highly similar to the migration of modern humans out of Africa and the environmental adaptation in modern Tibetans.

*Link of the paper: https://www.nature.com/articles/ncomms14458.

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Award Winners:

Junior Research Investigators Award Winner Dr. Hung-Yuan (Peter) Chi Deciphers DNA Repair Mechanism to Further Cancer Prevention and Treatment

Dr. Hung Yuan (Peter) Chi (冀宏源) received his PhD degree from Yale University, and joined the NTU Institute of Biochemical Sciences in 2010. His laboratory focuses on deciphering the mechanism of homologous recombination (HR)-mediated DNA repair. He hopes the molecular insights garnered from his studies could provide a basis for devising prevention and treatment strategies for cancers that arise from recombination repair deficiency. He received the Outstanding Teaching Award from NTU in 2014 and the Ta-You Wu Memorial Award from MOST in 2015.

Our human genome has suffered various forms of DNA damage from environmental carcinogens, high-energy ultraviolet light, and replication stress. HR represents a major error-free DNA repair pathway to eliminate pre-carcinogenic chromosomal lesions. Dr. Chi’s laboratory focuses on deciphering the action mechanism of recombination repair machinery. His recent studies have demonstrated that the repair protein, SWI5-SFR1 complex, has a dual function of promoting RAD51 recombinase activity to repair DNA double-strand breaks. Importantly, the physical protein-protein interaction between SWI5-SFR1 complex and RAD51 is a prerequisite for their repair function. Thus, Dr. Chi’s findings will expedite our understanding of the regulatory mechanisms of recombination repair at the molecular level.

The DNA repair mechanism identified in his studies could translate into precision medicine for the prevention and treatment of various cancers that arise from recombination repair deficiency. The most well-known example is the American actress, Angelina Jolie, who chose prophylactic surgery for cancer prevention after gene sequencing detected a mutation to a recombination repair gene like BRCA1. In addition, cancer cells with genetic mutations in recombination repair genes will be sensitive to specific drugs such as olaparib. Thus, his research in deciphering the repair mechanism has great potential and value for precision medicine applications.

College Student Research Creativity Award Winner Chia-Yi Lee Determines the Biochemical Properties of MCPH1 and Its Mechanistic Role in DNA Repair

Dr. Hung-Yuan (Peter) Chi from the Institute of Biochemical Sciences mentored his student, Chia-Yi Lee, in their award-winning college student research project that focuses on the biochemical properties of microcephaly-related protein MCPH1 and its mechanistic role in homologous recombination-mediated DNA double-strand break repair. Previous research indicated that deficiency in MCPH1 not only causes microcephaly but also leads to defects in DNA repair, which results in genome instability and might increase the risk of cancer. Moreover, MCPH1 can be co-immunoprecipitated with recombinase RAD51, which indicates the protein’s function in recombination repair. However, the molecular mechanism of MCPH1 remains unknown due to the difficulty of obtaining purified protein for functional analyses.

In their project, Dr. Chi and Lee successfully purified the full-length MCPH1 protein for the first time with the Expi293^TM mammalian cell expression system, and discovered its ability to bind single- and double-strand DNAs. Also, with purified MCPH1 and RAD51, they observed the direct interaction between MCPH1 and RAD51. On the basis of these findings, they ascertained MCPH1’s direct involvement in homologous recombination. Importantly, the biochemical characteristics of MCPH1 assist them to decipher the protein’s role in homologous recombination and microcephaly. Through their research in MCPH1, Dr. Chi and Lee have paved a road for the development of precision medicine for patients with cancer and microcephaly.

College Student Research Creativity Award Winner Jing-Yi Lu Unveils the Potential Effect of a Highly Efficient Generalized Brid Pollination System on Floral Evolution in East Asia

Associate Prof. Chun-Neng Wang (王俊能) from the Department of Life Science led students Kai-Hsiu Chen (陳凱修) and Jing-Yi Lu (盧璟誼) to investigate the pollination biology and pollination syndromes of the widespread Taiwanese Aeschynanthus acuminatus (Gesneriaceae). Funded by MOST’s College Student Participation in Research Projects, the research team conducted several field experiments and observations in Manyueyuan, Sanxia.

More than 160 sunbird-pollinated Aeschynanthus species are distributed in Southeast Asia. However, one species, A. acuminatus, has managed to extend its range beyond the distribution of its putative pollinators, sunbirds, in East Asia. The video recordings indicated that A. acuminatus has been successfully pollinated through generalist passerines, including the Taiwan yuhina (Yuhina brunneiceps) and the grey-cheeked fulvetta (Alcippe morrisonia), resulting in a moderately high natural fruit set (60%) and a high conspecific pollen transfer rate (93%). Generalist passerines have become efficient pollinators partly because the winter-blossoming A. acuminatus provides them with copious (60.52 μL) and highly diluted (6.8%) hexose-dominant nectars as seasonal diet during food shortage in winter. Also, the wide-open corolla tube and the reddish-green flower, the latter indicating the existence of a major peak in the long-wavelength reflectance spectrum, attract and allow generalist passerines to access nectars easily.

This study has made a major contribution to the research of the bird pollination system in East Asia, where birds other than sunbirds are rarely found as pollinators for flowering plants. The generalized bird pollination system of A. acuminatus provides new and strong evidences that generalist passerines not only act as efficient pollinators but also form a tight relationship with ornithophilous plants in the absence of specialist birds. This is the first detailed bird pollination study in Taiwan. It implies a potential role for generalist passerines to serve as winter pollinators and for the flowers of A. acuminatus to develop a corresponding bird pollination syndrome. Together, the birds and the flowers benefit from each other to evolve toward specialized plant-animal interactions.