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Analysis of sun and shade leaf formation in Arabidopsis thaliana

星野, 里奈 東京大学 DOI:10.15083/0002004517

2022.06.22

概要

植物は環境に応じて葉の厚さを可塑的に変えることが知られている。弱光下では薄い陰葉を形成する一方で、強光下では葉肉組織をさらに発達させた厚い陽葉を形成する。これらの生理的意義は数多く報告されてきたものの、その形成の分子メカニズムは未解明である。これは葉の厚さという観察しづらい形質に加え、これまで生理的解析に用いられた植物種では遺伝学的なアプローチが困難なためである。これらの問題点を克服するために、本研究ではシロイヌナズナを用いて陽葉と陰葉の形成過程を明らかにし、葉厚を制御する分子メカニズムの理解に向けて関連変異体の解析を行なった。

 初めに野生型のシロイヌナズナの葉原基を透明化し、組織断面の形態を定量した。陽葉は陰葉とは異なり、柵状組織における細胞伸長と並層方向の細胞分裂が、特定の期間に集中してみられることを発見した。この陽葉に特有な肥厚する過程を(A)極性方向成長、陰葉・陽葉共通の発生過程を(B)等方向成長と区分した。これら野生型の観察結果をもとに、厚い葉を形成することで知られているangustifolia変異体の葉の発生過程と比較した。angustifolia変異体では野生型の陽葉形成よりも早い時期から並層分裂を開始し、それを長期間続けることで葉が厚くなることがわかった。さらにangustifolia変異体は野生型とは異なり、葉の裏側にある海綿状組織でも並層分裂がみられた。これらの結果から、angustifolia変異体では野生型の陽葉形成とは別の仕組みによって葉が肥厚する可能性が示唆された。

 次に陽葉形成に重要な極性方向成長に影響する因子を特定するために、光を感知するタンパクである光受容体に着目し、それらが機能欠損した変異体を用いて陽葉形成への影響を解析した。その結果、青色光受容体phototropin(phot)とcryptochrome(cry)を全て欠失したcry1cry2phot1phot2四重変異体だけが、強光下でも薄い葉を形成することを発見した。また発生過程の観察により、この四重変異体では発生初期に見られた細胞伸長が徐々に失われていった結果、細胞が十分に伸長できないまま成熟し、葉が薄くなっていたことがわかった。つまりこの四重変異体の解析によって、陽葉形成には青色光シグナルに非依存的な発生初期の細胞伸長と、青色光シグナルに依存的な発生後期の細胞伸長との二通りの制御があることが示唆された。

 以上により、青色光受容体以外の経路も葉の肥厚に関わることが示唆されたため、別の因子にも着目した。ショ糖は強光下で合成量が高まり、かつ細胞分裂活性を促進させる作用があることから、ショ糖が及ぼす陽葉形成への影響を調べた。まず、ショ糖を添加した培地で野生型シロイヌナズナを栽培し、陰葉・陽葉形成への影響を評価した。その結果、ショ糖濃度が高まるにつれて葉の細胞総数が増加した一方で、葉の肥厚に寄与する葉断面の細胞層数や細胞伸長には影響しなかった。また、細胞サイズはショ糖濃度と光の強さの両作用によって増大することが、統計学的解析からも示唆された。以上から、糖は葉原基の発達時において細胞数を増加させ、かつ等方向成長を促すものの、極性方向成長でみられる並層分裂や細胞伸長には直接寄与しないことが判明した。

 本研究では、修士課程で発見した、強光下で核内倍化が促進する現象についても詳しく調べた。核内倍化と陽葉形成の因果関係や、強光下で核内倍化が促進する原因について調べるため、核内倍化が強く抑制されるbrassinosteroid insensitive 4(bin4)変異体を強光下で栽培した。その結果、弱光下に比べ強光下で栽培したbin4変異体は、核相がわずかに増加した。形態に関しては、等方向成長は強く抑制されたものの、極性方向成長には影響していなかった。さらに、このbin4変異体をショ糖入りの培地で栽培したところ、組織形態はほとんど変化しなかったものの、核相は弱光下でも強光下と同じレベルまで増加した。この結果から、核内倍化は陽葉の分化の必要条件ではないことがわかった。また、核内倍化の促進を引きおこす原因について、強光により合成されたショ糖による可能性が示唆された。これを確かめるため、野生型シロイヌナズナを異なるショ糖培地で栽培したところ、ショ糖濃度が高まるにつれて核相も高まることを発見した。同様に青色光受容体を欠損したcry1 cry2 phot1 phot2四重変異体をショ糖培地で栽培すると、ショ糖による核内倍化の促進作用が完全に抑制されていた。その一方で、四重変異体の細胞サイズや細胞伸長はショ糖により若干回復することがわかった。これらの結果から、①核内倍化は等方向成長との相関がみられるが、極性方向成長には寄与しないこと、②強光下での核内倍化は青色光シグナルやショ糖により制御されていることがわかった。

 本研究を通じて、陽葉の肥厚は多段階的な発生過程を経ること、及びそれらの過程は異なる誘導因子により、異なるタイミングで引きおこされることがわかった。そして、それらの誘導には青色光シグナルやショ糖が関与することがわかった。さらに、強光下では青色光受容体とショ糖によって核内倍化が促進される一方で、核内倍化の促進自体は陰葉・陽葉の分化に寄与しないことが新たに示唆された。

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