Research of Hiro's Lab

Plants have evolved unique adaptation systems to tolerate various environmental stresses. An understanding of the fundamental mechanisms underlying adaptation processes is expected to provide novel ideas for improving plant functions. The main goal of my goup is to dissect plant specific signaling networks essential for the adaptation system by developing and employing advanced proteomics technologies.

Currently, we are focusing on establishing proteomics methods to analyze phosphorylation events in plants. By utilizing the established methods, we are attempting to dissect plant immune siganling pathways.

Generally, plants recognize environmental changes and transmit signals using protein factors, which lead to gene expression and metabolic changes for adaptation. Post-translational modifications (PTMs) are often utilized as a means for the proteins to transmit signals to downstream events. Among various PTMs, ‘phosphorylation’ is known to play a vital role in regulating all sorts of cellular phenomena and as a mediator of signaling pathways in plant cells. The function of this process in regulating plant growth and development in particular makes it highly attractive for plant engineering. Therefore, development of phosphoproteomics technologies, which can monitor phosphorylation status of cellular proteins comprehensively, have been expected to be powerful tools to unravel novel signaling pathways especially involved in early responses.

We have established a post-translational modification (PTM)-oriented liquid chromatography- mass spectrometry (LC-MS) based shotgun proteomics platform primarily focused on phosphorylation. One of the key requirements for successful PTM-oriented proteomics is the establishment of efficient enrichment methods for post-translationally modified peptides or proteins. In the first report of its kind, we successfully adapted phosphopeptide enrichment methods, which have been developed for other organisms, to plant materials, and thereby succeeded in providing an overview of the events occurring during in vivo phosphorylation in plants (Arabidopsis) at the cellular level (Publication #1).

Transferring information from model plant species such as Arabidopsis to other plant species still remains a considerable challenge. In particular, whether information on protein modifications from model plants is readily applicable to evolutionarily divergent plant species remains unclear because of limited evidence about whether conserved residues are modified in the same manner. To investigate the conservation of phosphoproteomes between plant species, we further collected phosphoproteome data from rice cells and performed a comparative analysis of rice and Arabidopsis. Our analyses identified a large number of conserved phosphorylation sites in rice and Arabidopsis and provided the first overview of phosphoproteome conservation between dicots and monocots (Publication #2).

The infection of plants by pathogens is causing considerable economic loss to agricultural industries. An understanding of the fundamental mechanisms underlying disease resistance processes is expected to provide novel ideas for improving the current status.

The extensive analyses conducted previously were largely based on forward genetics, and the findings revealed that plants utilize a two-branched innate immune system for defense against pathogens. In the first branch, transmembrane pattern recognition receptors (PRRs) are used to recognize and respond to slowly evolving microbe-associated molecular patterns (MAMPs). In the second branch, either direct or indirect recognition of the pathogen through disease-resistance (R) proteins is used for response to pathogen virulence factors. Knowledge regarding pathogen recognition mechanisms has been accumulated rapidly, but information on the molecular mechanisms underlying the signal transduction that leads to defense responses is thus far limited.

The poor outcomes of research on plant immune signaling mostly stem from the limitations of forward genetics. With the forward genetics approach, it is difficult to isolate the plant immunity-related genes that are also essential for plant development or belong to functionally redundant gene families. To overcome this problem, we are applying advanced proteomics technologies in the identification of novel factors that regulate plant immune siganling pathways.

Phosphorylation is critical for the activation and functioning of most proteins, and it has been shown to regulate plant immunity. Importantly, identified PRRs or their interactors were found to be receptor kinases. Therefore, differential phosphoproteomics can be an effective breakthrough approach in understanding immune signaling pathways in plants and in identifying novel targets for molecular breeding in order to benefit agricultural industries.

Protein factors that are differentially phosphorylated in response to pathogen recognition can be isolated by differential phosphoproteomics. This approach is expected to result in not only the isolation of novel factors that regulate plant immunity but also the identification of regulatory phosphorylation sites corresponding to known factors, which will help in further investigation of protein functions.

Phosphoproteome dynamics in response to plant immune stimulants, shuch as MAMPs and plant hormones, will be analyzed by our advanced proteomics platform. Molecular functions of the identified phospho-regulated factors will be further analyzed to understand plant immune signaling pathways.

• A Brief History of Marchantia from Greece to Genomics
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In collaboration with bioinformatics specialists, we have developed a RIPP-DB (RIKEN Phosphoproteome Database), which provides information on phosphopeptides identified from Arabidopsis and rice. We also contributed to develop a proteomics aggregation utility called the MASCP Gator, which can view the phosphopeptide information kept in the RIPP-DB (Publication #3).