Heterotrimetic G proteins (consisting of subunits Gα, Gβ, and Gγ) are signaling molecules widely found in eukaryotic organisms. In plants, G proteins play roles in signal transduction of phytohormones such as auxin and abscisic acid (ABA). Molecular mechanisms of G protein-mediated signaling have been well characterized in animals, but the numbers and kinds of components of G protein-mediated signaling are different between animals and plants, raising the possibility that plants have plant-specific mechanisms for G protein-mediated signaling. Here, studies on Arabidopsis thaliana Gβ (AGB1) and its interaction partners are described. The AGB1-interaction partners focused on are: (1) signal transduction of a phytohormone, brassinosteroid (BR); (2) VIP1, a bZIP protein which physically interacts with AGB1.

1. AGB1 regulates brassinosterid signaling independently of BZR1

An AGB1-null mutant, agb1-2, is known to be hyposensitive to BR in seed germination. Moreover, both agb1-2 and another AGB1-null mutant, agb1-1, have the phenotypes similar to the phenotypes of mutants that have defects in BR signaling (e.g. rounder leaves and higher ABA sensitivities). An established model of BR signaling consists of specific types of protein kinases, protein phosphatases, and transcription factors, but it is still unclear whether AGB1 interacts with those components of BR signaling.

A key transcription factor downstream of BR, BZR1, and its gain-of-function mutant, bzr1-1, were overexpressed in an AGB1-null mutant, agb1-1, to examine their effects on the agb1 phenotypes, and to examine whether AGB1 regulates the functions of BZR1. Because the amino acid sequence of AGB1 contains 17 putative modification motifs of Glycogen synthase kinase 3/SHAGGY-like protein kinases (GSKs), which phosphorylate BZR1 and thereby regulate BR signaling, the interaction between AGB1 and one of Arabidopsis GSKs, BIN2, was examined.

Expression of bzr1-1 alleviated the inhibitory effects of a BR biosynthesis inhibitor, brassinazole, on hypocotyls elongation in both wild type (WT) and agb1-1. Overexpression of BZR1 alleviated the inhibitory effects of ABA on cotyledon greening in both WT and agb1-1. AGB1 interacted with BIN2 in vitro. However, AGB1 did not affect the phosphorylation state of BIN2, and was not phosphorylated by BIN2 in vitro. AGB1 did not affect the phosphorylation state of BZR1 in vivo, either. These results suggest that AGB1 interacts with BIN2, but regulates BR signaling in a BZR1-independent manner.

2. Studies on an AGB1-interacting protein, VIP1

2.1. VIP1 is a regulator of osmosensory signaling

A yeast two-hybrid (Y2H) screen using the Arabidopsis leaf cDNA library identified a bZIP protein, VIP1, as a potential AGB1-interacting protein. During an investigation of the subcellular localization of VIP1, it was discovered that the nuclear localization of VIP1 is rapidly enhanced in response to rehydration. This interesting finding tempted the author to examine the interaction between VIP1 and two ABA-inactivating genes, CYP707A1 and CYP707A3 (CYP707A1/3), which are known to be induced by rehydration.

The subcellular localization of VIP1 was examined by expressing GFP-fused VIP1 (VIP1-GFP) in Arabidopsis. The interactions between VIP1 and CYP707A1/3 promoters were examined by a transient reporter assay, gel shift assays, and chromatin immunoprecipitation (ChIP). The expressions of CYP707A1/3 in a VIP1-null mutant, vip1, and VIP1-GFP-expressing plants were also examined.

VIP1-GFP signals in the nucleus were enhanced within 10 minutes, and then gradually weakened when plants were submerged in a hypotonic solution. The time course of the changes of the subcellular localization of VIP1-GFP was consistent with the time course of CYP707A1/3 inductions during rehydration. The transient reporter assay, the gel shift assays, and the ChIP assay all showed that VIP1 interacts with the CYP707A1/3 promoters, supporting the idea that VIP1 regulates the CYP707A1/3 expressions in osmosensory signaling. The gel shift assays suggested that AGCTGT/G are at least partially responsible for the interaction between VIP1 and the CYP707A1/3 promoters. The expression level of CYP707A1 was lowest in vip1 and highest in VIP1-GFP-expressing plants, but no clear difference was observed in the expression level of CYP707A3 among the genotypes studied, raising the possibility that VIP1 and its close homologs are functionally redundant.

2.2. VIP1 interacts with AGB1

The interaction between AGB1 and VIP1 was further examined by Y2H assays, an in vitro GST pull-down assay, and bimolecular fluorescence complementation (BiFC). Because VIP1 was suggested to play a role in osmosensory signaling, effects of extracellular osmolarities on the AGB1-VIP1 complex were examined by a BiFC assay using onion epidermal cells. VIP1-GFP was expressed in agb1-1 to examine the effects of AGB1 on the functions of VIP1.

In the Y2H assays and the GST pull-down assay, both full-length VIP1 and the C terminal region (amino acids 165-341) of VIP1 interacted with AGB1. BiFC signals were speckled when transformed cells were incubated in distilled water, but not when cells were incubated in 0.5 M mannitol, suggesting that extracellular osmolarities regulate the subcellular localization and/or formation of the AGB1-VIP1 complex. However, no clear difference was observed between WT and agb1-1 in either the time course of the nuclear-cytoplasmic shuttling of VIP1 or the expression levels of CYP707A1/3 and another VIP1 target gene, MYB44. Expression of VIP1-GFP did not significantly affect the ABA hypersensitivity of agb1-1, either. Thus, although AGB1 and VIP1 physically interact, the physiological relevance of the interaction between AGB1 and VIP1 is still unclear.

2.3. Further studies on VIP1

Further studies on VIP1 were performed to test the possibilities that (1) rehydration triggers changes in the modification pattern and/or the conformation of VIP1, and that (2) VIP1 and its close homologs are functionally redundant.

Because phosphorylation is a possible modification of VIP1, the phosphorylation state of VIP1 during rehydration was examined using Phos-tag SDS-PAGE. This experiment suggested that VIP1 is dephosphorylated when its nuclear localization is enhanced upon rehydration. Okadaic acid, an inhibitor of type 1 and type 2A protein phosphatases (PP1 and PP2A, respectively), inhibited the rehydration-induced enhancement of the nuclear localization of VIP1. Components of Arabidopsis PP1 and PP2A were subjected to a Y2H analyses, and ATB'κ, one of the B' subunits of PP2A, was found to interact with VIP1. These results strongly support the possibility (1), namely the idea that phosphorylation is involved in regulating the subcellular localization of VIP1. It is also possible that factors other than phosphorylation regulate the subcellular localization of VIP1.

Quantitative RT-PCR showed that some of the genes encoding close homologs of VIP1 (plant group I bZIP proteins) are expressed as highly as VIP1. Two of the group I bZIP proteins, PosF21 and bZIP29, were expressed as GFP-fused proteins in Arabidopsis, and both showed the rehydration-responsive nuclear-cytoplasmic shuttling as VIP1 showed. Y2H and BiFC experiments suggested that VIP1 can form a homodimer and heterodimers with other bZIP proteins. Together, these results support the possibility (2).

Here the relation between AGB1 and BR signaling has been addressed in more details, and VIP1 has been identified as a novel regulator of osmosensory signaling. However, many points remain unclear. Further studies are required to elucidate the molecular mechanisms underlying the AGB1-mediated signaling pathway and the VIP1-mediated signaling pathway, and to determine how the two signaling pathways interact with each other.

三量体Gタンパク質(以下Gタンパク質。Gα, Gβ, Gγのサブユニットから構成される)は真核生物に保存された、細胞内信号伝達に関わるタンパク質である。Gタンパク質に関する研究は動物において先行しているが、動植物間でのゲノム情報を比較すると、Gタンパク質介在性信号伝達に関わる因子の数や種類には違いが見られる。このことから、よく調べられた動物のものとは異なる、植物独自のGタンパク質介在性信号伝達の機構が存在する可能性が想定される。シロイヌナズナのGβサブユニット(AGB1)の欠損変異体は野生型との表現型の違いが顕著であり、AGB1は重要な生理機能を持つと考えられる。このことから申請者はAGB1及びその相互作用因子に着目して解析を行い、植物のGタンパク質介在性信号伝達経路の一端を解明することを目指した。

1章の緒論では、研究の背景、意義と目的について述べた。

2章では、ブラシノステロイド(BR。植物ホルモンの一つ)の信号伝達経路に対するAGB1の関与について検討した。

AGB1の欠損変異体の表現型からAGB1のBR応答性への関与は示唆されていたものの、確立されたBR信号伝達のモデルは、特定の型のプロテインキナーゼ・ホスファターゼによるリン酸化のリレーとそれによる下流のBZRファミリーなる転写因子のリン酸化状態の制御により構成されており、AGB1がこれら既知のBR関連因子を制御するか否かは不明であった。

BR生合成阻害剤のブラシナゾール(BRZ)を用いた解析等により、AGB1の欠損変異体であるagb1-1とagb1-2のBR感受性低下を確認した。そして、BZR1なるBR応答経路の下流の転写因子と、それに点変異を持つbzr1-1をagb1-1に過剰発現させた。野生型におけるbzr1-1の発現はBRZに対する耐性を付与することが知られていたが、bzr1-1の過剰発現によりagb1-1の遺伝的背景においてもBRZの影響が低減された。また、BZR1の過剰発現によりABAの影響が低減されることも見出した。BRZとABAへの応答性に対して、BZR1又はbzr1-1過剰発現の効果とAGB1の欠損の効果は相加的であったことや、BZR1のリン酸化状態及び細胞内局在の解析から、AGB1はBZR1と独立にBR応答を制御することが示唆された。

GSKなる一群のプロテインキナーゼも既知のBR応答経路の制御因子である。AGB1のアミノ酸配列には17の推定GSK修飾サイトがあり、それらの幾つかはAGB1の推定立体構造の表面に位置したため、GSKの一つであるBIN2とAGB1との相互作用を解析した。この結果、生理的意義は不明であるものの、AGB1とBIN2がin vitroで相互作用することが明らかになった。

3章では、AGB1の物理的相互作用因子であるbZIPタンパク質VIP1について詳細な解析を行った。

酵母ツーハイブリッド法によりAGB1の物理的相互作用因子を探索し、VIP1が酵母細胞内でAGB1と相互作用することを見出した。更に、プルダウン解析と二分子蛍光相補法(BiFC)により、in vitro及び植物細胞でのAGB1とVIP1との相互作用を確認した。この相互作用の生理的意義に関しては不明な点が多く残ったが、BiFCのパターンから、VIP1の浸透圧応答経路における機能(後述)をAGB1が制御する可能性が示唆された。

次にVIP1の機能に関して詳細に解析を行い、細胞の吸水に伴う膨圧の上昇時にVIP1の核局在性が一過的に増大することを見出した。CYP707A1とCYP707A3(CYP707A1/3)なる遺伝子は細胞の吸水に応じて一過的にその転写量が増大することが知られており、その転写量の経時変化が吸水時のVIP1の細胞内局在の経時変化と整合すると考えられたため、VIP1とCYP707A1/3との相互作用について解析を行った。この結果、VIP1はin vitro、in vivoでCYP707A1/3のプロモーター領域に結合し、そのプロモーター活性を上昇させうることが示された。また、VIP1の過剰発現により高浸透圧ストレス下での成育遅延が促進されることも明らかになり、これらの結果よりVIP1の浸透圧応答経路への関与が強く示唆された。また、VIP1のリン酸化状態を解析し、PP1又はPP2Aなるプロテインホスファターゼによる脱リン酸化がVIP1の核局在性増大に必要であることを示した。更に、複数のVIP1相同因子に関しても解析を行い、それらをコードする遺伝子がVIP1と同程度に転写されていること、それらの細胞内局在がVIP1と同様の挙動を示すこと、それらがVIP1と二量体を形成しうることを確認し、VIP1とVIP1相同因子が浸透圧応答経路において冗長に機能する可能性を示した。

以上のように、本研究ではヘテロ三量体Gタンパク質とその物理的相互作用因子について詳細な解析を行い、ブラシノステロイド信号伝達へのAGB1の関与と、AGB1の相互作用因子VIP1の機能、特に植物の浸透圧応答経路に対する関与について極めて興味深い結果を得た。本研究で得られた成果は、植物の環境応答を理解する上で非常に重要であり、学術上、応用上貢献することが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。