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Molecular genetic studies on stem cell maintenance in Oryza sativa

鈴木, 千絵 東京大学 DOI:10.15083/0002001913

2021.10.04

概要

【序論】
植物は胚発生の後も持続的に器官を分化させ、生涯を通じた成長を示す。これは未分化な細胞集団からなる分裂組織「メリステム」の働きによるものである。メリステムでは常に幹細胞が維持されており、幹細胞が増殖することで器官分化に必要な細胞群が生み出される。すなわち、メリステムにおける幹細胞維持は、植物の持続的な発生の要であるといえる。
 真正双子葉植物シロイヌナズナの茎頂メリステムでは、低分子ペプチドCLAVATA3(CLV3)をリガンドとするシグナル伝達経路およびhomeobox型転写因子WUSCHEL(WUS)による負のフィードバックループによって幹細胞が維持されている。このCLV-WUSシグナリングに関与する因子は複数報告されているものの、植物の幹細胞維持を司る分子機構については未だ不明点が多い。
 当研究室の先行研究により、単子葉植物イネではCLV3のオルソログであるFLORAL ORGAN NUMBER2(FON2)が幹細胞増殖を負に制御することが明らかとなっている。本研究では、fon2亢進変異体(1B-280)に着目することで幹細胞維持に関与する新規因子の単離および機能解析を行い、植物の幹細胞維持機構に関する新規知見を得ることを目指した。

【結果】
1. Aberrant spikelet and panicle 1(asp1)変異はfon2変異を亢進する
1B-280は、機能欠損変異体であるfon2-3に変異原処理を行って得られたM2集団中より見出された。fon2-3では、幹細胞が過剰に増殖することが原因と考えられる花器官数の増加が観察されるが、1B-280ではその異常が亢進されていた。これは、1B-280が有する第二の変異がFON2と機能的に関連する因子に生じている可能性を示唆している。
 原因遺伝子の同定を試みたところ、1B-280ではABERRANT SPIKELET AND PANICLE1(ASP1)遺伝子に重篤な変異が生じていることが明らかとなった。1B-280に機能的なASP1遺伝子を導入すると、fon2変異の亢進が抑圧されたことから、1B-280においてfon2変異を亢進する原因がasp1変異であることが示された。以後、1B-280をfon2-3 asp1-fe(fe; fon2 enhancer)と表記する。

2. ASP1は幹細胞増殖を負に制御する
ASP1はシロイヌナズナTOPLESSのホモログであり、Groucho/Tup1型転写コリプレッサーをコードする。ASP1が花序形態制御等に関与することは報告されているものの、幹細胞維持における機能は未知である。ASP1の幹細胞維持における機能を明らかにするため、各変異体のメリステムにおけるFON2mRNAの発現パターンを解析した。FON2は、メリステムの幹細胞に相当する領域で発現する。fon2-3ではFON2mRNA領域の拡大が見られたが、asp1では野生型と同様の発現パターンが観察された。一方、fon2-3asp1-feではFON2の発現領域が著しく拡大している様子が観察された。これらの結果は、fon2変異による幹細胞増加の表現型がasp1変異によって相乗的に亢進されることを示し、ASP1がFON2の機能と密接に関連しつつ幹細胞増殖を抑制する機能を持つ可能性を示唆している。
 また、ASP1の発現パターンを解析したところ、FON2発現領域と重なる部分で発現が認められた。これは、ASP1がFON2と協調しながら幹細胞維持に機能する可能性を支持する結果であると考えられる。

3. ASP1はFONシグナリングと共通する遺伝子群の発現を制御することにより幹細胞増殖を調整する
幹細胞増殖の抑制におけるASP1の分子機能を明らかにするため、マイクロアレイ解析を行った。野生型と比較してfon2, asp1, fon2 asp1で発現が変動していた遺伝子を抽出したところ、asp1で発現が変動していた遺伝子の多くがfon2においても発現変動することが明らかとなった。これは、ASP1によって制御される遺伝子の多くがFONシグナリングによっても制御されることを示す。
 さらに、得られた発現変動遺伝子群についてGene Ontology(GO)解析を行ったところ、fon2で発現上昇した遺伝子群で検出されたGOの多くが、fon2 asp1で発現上昇した遺伝子群でより有意に検出された。これは、表現型で観察されていたasp1変異によるfon2変異の亢進を遺伝子発現レベルで示す結果であり、ASP1とFONシグナリングが抑制する遺伝子群の機能が類似していることを示唆している。

【考察】
本研究では、ASP1がFONシグナリングと協調しながら幹細胞維持に機能することを明らかにした。ASP1はFONシグナリングと共通した遺伝子群の発現を制御することが示されたが、asp1変異はfon2変異を表現型および遺伝子発現レベルで相乗的に亢進する効果を持っていた。これは、ASP1が幹細胞維持においてFONシグナリングと類似した作用を持ちつつも、完全に同一の経路では機能しない可能性を示唆している。
 一般に、CLV-WUSシグナリングは幹細胞数を一定に制御するfine-tuning機構であると捉えられており、イネのFONシグナリングも同様であると考えられる。ASP1は転写コリプレッサーとして、幹細胞維持に機能する多数の遺伝子の発現を適切に抑制することで、FONシグナリングによるfine-tuningを容易とする機能を持つ可能性が考えられる。

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