( 主査 ) 教授髙橋秀幸教授山口信次郎准教授佐藤修正

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2 氏名 ( 本籍地 ) 学 位 の 種 類 学 位 記 番 号 学位授与年月日 学位授与の要件 研究科, 専攻 論 文 題 目 ぱんれい 庞磊博士 ( 生命科学 ) 生博第 337 号平成 29 年 3 月 24 日学位規則第 4 条第 1 項該当東北大学大学院生命科学研究科 ( 博士課程 ) 生態システム生命科学専攻 Analyses of Functional Tissues and Hormonal Regulation for Hydrotropism in Arabidopsis Roots ( シロイヌナズナ根の水分屈性に機能する組織およびホルモン制御の解析 ) 博士論文審査委員 ( 主査 ) 教授髙橋秀幸教授山口信次郎准教授佐藤修正

3 論文内容の要旨 GNOM/MIZ2 could function in multiple tissues such as epidermis, cortex and stele for both hydrotropism and phototropism. Above-mentioned results suggested that MIZ1 and GNOM/MIZ2 function in tissues other than the root cap and/or the meristem. I therefore conducted surgical experiments with Arabidopsis roots and found that removal of the root cap resulted in significant reduction of gravitropic response, but not of hydrotropic one. Furthermore, ablation of both the root cap and the meristem with micro-beam laser did inhibit gravitropic response but not hydrotropic response in the hydrotropism-rescued lines of the gnom/miz2 mutant (Fig. 2). Thus, the results in this study implied that MIZ1 functions in the cortex of the transition and/or elongation zones and that both sensing of and response to moisture gradients occur in the transition and/or elongation zones of Arabidopsis roots. Also, both MIZ1 and GNOM/MIZ2 could play a role in phototropic signaling at the transition and elongation zones of Arabidopsis roots. Figure 1. Expression of MIZ1-GFP in cortex is responsible for complementing the hydrotropic defect of miz1 roots (a,b) Expression pattern of MIZ1-GFP fusion protein under control of the WER (a) and PIN2 (b) promoters with HSP terminator. Arrowhead indicates the boundary between transition and elongation zones. Scale bar indicates 100 µm. (c) Hydrotropic curvature of Col-0, miz1, WER:MIZ1-GFP-HSPter and PIN2:MIZ1-GFP-HSPter plants 12 h after transfer of seedlings to the moisture gradient in air assay system. Data are mean ± SE (n = 35-44) of three independent experiments. Different letters indicate statistically significant differences (p < 0.05, Tukey HSD test). (d-g) Representative images of hydrotropic response in roots of Col-0 (d), miz1 (e), WER:MIZ1-GFP-HSPter (f) and PIN2:MIZ1-GFP-HSPter (g) after 12 h of hydrostimulation. Scale bar indicates 1 mm.

4 Figure 2. Ablation of both the root cap and the meristem with micro-beam laser does not inhibit hydrotropism (a-e) Confocal images of propidium iodide (PI)-stained primary root tip after the laser ablation. (a) Col-0; (b) RCH1:GNOM-GFP; (c) Co2:GNOM-GFP; (d) WER:GNOM-GFP; (e) SHR:GNOM-GFP. Arrowhead indicates the boundary between elongation and transition zones. Scale bars indicate 200 µm. Hydrotropic curvature (f) and growth (g) of roots of 4-day-old seedlings treated with or without laser ablation after 8 h of hydrostimulation using the moisture gradients in air assay. Data are mean ± SE from three or four independent experiments (n = 16-28). Different letters indicate statistically significant differences (p < 0.05, Tukey HSD test). 2. Hormonal regulation of hydrotropism in Arabidopsis roots Plant hormones are implicated to regulate root responses to environmental stimuli. Auxin polar transport plays a critical role in inducing gravitropic response, whereas it is not required for hydrotropism in Arabidopsis roots. On the other hand, it is considered that ABA positively regulates root hydrotropism. Besides, a possible involvement of cytokinin in hydrotropism has been proposed. However, the mechanisms for hormonal regulation of hydrotropism are poorly understood. To address this question, I investigated the regulatory mechanisms of auxin- and cytokinin-mediated hydrotropism in relation to ABA regulation of hydrotropism. First, I used direct auxin sensor DII-VENUS to monitor endogenous auxin level and demonstrated that hydrotropic response was accompanied by a reduction of endogenous auxin content in the meristem zone. This decrease of auxin content was substantially repressed by miz1 mutation. In addition, pharmacologically lowering auxin content with an inhibitor of

5 auxin biosynthesis, L-Kynurenine (KYN), significantly accelerated hydrotropism, although it substantially reduced gravitropism. Clinorotation of Arabidopsis seedlings in the presence of moisture gradients enhanced hydrotropic response, but KYN treatment did not further enhance the hydrotropic response of clinorotated roots. Considering the counteraction between gravitropism and hydrotropism, it was assumed that KNY-enhanced hydrotropism was due to the attenuated gravitropism. It was found that not only auxin but also endogenous cytokinin content decreased in the root tip in response to moisture gradients. Namely, hydrostimulation resulted in a decrease in activity of cytokinin reporter ARR5::GFP and in transcript level of cytokinin biosynthesis IPT genes. Exogenously supplementing cytokinin remarkably inhibited hydrotropism, while mutations in IPT genes and cytokinin receptor AHK genes accelerated hydrotropism. These results suggested that cytokinin acts as a negative regulator of hydrotropism. However, the reduction of cytokinin content was not repressed by miz1 mutation. Thus, MIZ1 could be involved in the regulation of auxin content but not of cytokinin. Next, I examined whether the inhibition of hydrotropism by cytokinin and auxin is attributable to antagonizing ABA response. Application of exogenous cytokinin or auxin inhibited hydrotropism of wild type roots, but not of snrk2.2 snrk2.3 double mutant (Fig. 3). Moreover, application of exogenous ABA could relieve the cytokinin- or auxin-repressed hydrotropism in wild type roots. Furthermore, treatment with cytokinin and auxin together caused repression in hydrotropism of snrk2.2 snrk2.3 double mutant (Fig. 3). These observations indicated that cytokinin and auxin antagonistically function in ABA-inducible hydrotropism and that cytokinin and auxin synergistically repress hydrotropic response. Figure 3. Effects of cytokinin and auxin on ABA-inducible hydrotropism Hydrotropic response (a) and root growth (b) of Col-0 and snrk2.2 snrk2.3 double mutant treated with cytokinin or auxin, or cytokinin and auxin together (n = 27-43). Data are mean ± SE from three independent experiments. Different letters indicate statistically significant differences (p < 0.05, Tukey HSD test). 3. A proposed model for hydrotropism in Arabidopsis roots Based on the results of this study, I proposed a novel mechanism for hydrotropism in Arabidopsis

6 roots. Perception of hydrotropic stimulation occurs in the transition and/or elongation zones but not in the root cap or meristem. MIZ1 functions in cortex of the transition and/or elongation zones, while GNOM/MIZ2 functions in epidermis, cortex or stele of the transition and/or elongation zones (Fig. 4). Furthermore, hydrotropic response is accompanied by a reduction of endogenous auxin and cytokinin contents in the root tip, which could diminish the counteraction from root gravitropism and enhance the ABA-mediated hydrotropism (Fig. 5). Thus, the molecular mechanism for hydrotropism is distinct from that for gravitropism in Arabidopsis roots. Figure 4. Schematic model for MIZ1- and GNOM/MIZ2-regulalted hydrotropism in Arabidopsis roots Perception of moisture gradient does not require root cap or meristem, but takes place at the transition (TZ) and/or elongation zones (EZ) where the hydrotropic bending occurs. MIZ1 (yellow dots) functions in cortex of the transition and/or elongation zones, while GNOM/MIZ2 (red dots) functions in epidermis, cortex or stele of the transition and/or elongation zones for the induction of hydrotropic response in Arabidopsis roots. Figure 5. Schematic model for hormonal regulation of hydrotropism in Arabidopsis roots Roots reduce endogenous cytokinin and auxin contents and accumulate ABA level in response to moisture gradients. Cytokinin or auxin primarily antagonizes SnRK2-depdendent ABA signaling and inhibits hydrotropism. Thus, reduction of auxin and cytokinin contents could, at least in part, attenuate the counteractive interaction from gravitropism, elevate ABA response, and facilitate hydrotropic response in Arabidopsis roots.

7 論文審査結果の要旨 植物の根は 重力屈性および水分屈性を発現させて 高水分側に伸長し 乾燥ストレスを軽減 回避する 根の重力屈性では 根冠のコルメラ細胞が重力刺激を感受し 植物ホルモンであるオーキシンの輸送体の局在が変化し それによって形成されるオーキシンの伸長帯における不均等分布が屈曲成長を誘導する 一方 根の水分屈性の分子機構はよく理解されていない とくに 水分屈性に必須な MIZ1 および MIZ2 は水分屈性に特異的に機能するが その分子機能は未解明のままである また 水分勾配の感受機構や 水分屈性における植物ホルモンの作用についても不明な点が多い そこで Pang Lei 氏は シロイヌナズナの根の水分屈性で MIZ1 および MIZ2 が機能する細胞群を同定し さらに 水分屈性に対する植物ホルモンの作用様式を解析し 根の水分屈性と重力屈性の分子機構を比較した まず 組織特異的プロモーターを用いて 水分屈性を欠損した miz1 miz2 突然変異体の根の異なる組織に MIZ1-GFP および MIZ2-GFP を発現させ それぞれのタンパク質の発現部位と水分屈性の回復の関係を解析した その結果 miz1 突然変異体の水分屈性は MIZ1-GFP を根の初期伸長領域 伸長帯の皮層細胞に発現させた場合に回復することを発見した miz2 突然変異体の水分屈性は MIZ2-GFP を初期伸長領域 伸長帯の表皮 皮層 中心柱に発現させた場合に回復した さらに レーザーアブレーション法で根冠および分裂組織を破壊しても水分屈性は正常に発現することを確認した これらの結果は 根の水分屈性に MIZ1 の皮層特的発現を必要とすること 水分勾配の感受と偏差成長 ( 屈曲 ) が根の初期伸長領域 伸長帯で生ずることを明らかにした 次に 根の水分屈性におけるオーキシンとサイトカイニンの役割を それらの応答遺伝子をマーカーとして解析し オーキシンおよびサイトカイニンが水分屈性を負に調節すること その場合 オーキシンは重力屈性を制御することによって水分屈性に間接的に影響し サイトカイニンはアブシジン酸応答を担う SnRK2 に働き MIZ1 の発現量を低下させる可能性を示した これらの結果は ホルモン制御が水分屈性と重力屈性で異なることを明らかにした 以上の成果は 根の水分屈性のユニークな分子機構の理解に貢献するもので Pang Lei 氏が自立して研究活動を行うに必要な高度の研究能力と学識を有することを示している したがって Pang Lei 氏によって提出された論文は 博士 ( 生命科学 ) の博士論文として合格と認める