To escape low oxygen conditions, numerous adaptations at the anatomical, morphological and metabolic level took place in plants suffering from waterlogged conditions. One of the structural adaptations is aerenchyma formation. Aerenchyma is a tissue consisting of longitudinal gas spaces separated by the strands of living cells, found in the root cortex of waterlogged plants. It enables the passage of gases in and out of tissues in plant roots. Internal transport of oxygen via the aerenchyma from shoots to roots is especially important for survival under waterlogged conditions. However, the molecular mechanism of lysigenous aerenchyma formation remains to be elucidated. The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation.

1.Root growth and aerenchyma formation of maize under waterlogged or aerobic conditions

In this section, the growths of roots and shoots and aerenchyma formation were investigated. Three-day-old seedlings were treated under waterlogged conditions for 48 hours (h). The growth of the roots was retarded under waterlogged conditions compared with the roots grown under aerobic conditions. No effect was found of the waterlogged treatment on the elongation of the shoots. Three-day-old aerobically grown seedlings were then subjected to waterlogging treatment for 48 h and aerenchyma formation was investigated at different levels at the basal part of the root. I found that the gas spaces increased significantly during the treatment and the most inducible part was at 1.5-2cm from the root-shoot junction.

2. Identification of genes involved in aerenchyma formation in maize roots using a laser microdissection and microarray analysis

Ethylene is known to be involved in aerenchyma formation in maize roots. It has been reported that aerenchyma is formed through programmed cell death (PCD). Three-day-old aerobically grown seedlings were then treated with ethylene (1ppm) under aerobic conditions. The result showed that ethylene induced aerenchyma formation, which started between 6 h and 12 h after the ethylene treatment. On the other hand, aerenchyma formation was not observed at the basal region of roots of 4-day-old seedlings grown under aerobic conditions. The use of 1-methylcyclopropene (1-MCP; 1ppm), an inhibitor of ethylene perception, completely blocked the formation of aerenchyma in the maize cortical cells when treated under waterlogged conditions for 24 h. Whereas the treatment of seedlings under waterlogged conditions without the 1-MCP pre-treatment induced aerenchyma formation starting between 18 h and 24 h after the treatment. Because aerenchyma is formed in the cortical cells of the root, cortical cells of roots treated under waterlogged conditions with or without pre-treatment with 1-MCP or aerobic conditions were isolated by laser microdissection and their mRNA levels were examined with a microarray. As a result, the signal intensities of 575 genes among 42,034 gene probes spotted on a microarray slide were significantly different between the two conditions for inducing and not inducing aerenchyma formation. Among them, it was likely that 239 genes were up-regulated and 336 genes were down-regulated under conditions for inducing aerenchyma formation (waterlogged conditions). The differentially expressed genes included genes related to generation or scavenging of reactive oxygen species (ROS), cell wall loosening and degradation, and Ca2+ signaling.

3. Confirmation of microarray result using a semi quantitative RT-PCR and analysis of tissue specificity of the selected genes

Production of ROS has been implicated in diverse physiological processes including PCD in plants. One of major sources of ROS in plants is a reaction mediated by NADPH oxidase, which is responsible for the conversion of O2 to superoxide anion (O2), thereby leading to production of hydrogen peroxide (H2O2). Several ethylene-mediated waterlogging-responsive genes related to ROS-generation or ROS-scavenging were found. The up-regulated genes contain gene encoding respiratory burst oxidase homolog (RBOH) and the down-regulated genes contain gene encoding metallothionein (MT). The RBOH gene is involved in ROS generation and the MT gene is involved in ROS scavenging. To determine the tissue specificity of these genes, the stelar cells and the cortical cells of maize roots, treated or not under waterlogged conditions, were collected separately using the laser microdissection and the expression of these genes were analyzed using a semi quantitative RT-PCR. The up-regulation of RBOH expression was observed in both cortical cells and stelar cells, but the mRNA levels appeared to be slightly higher in cortical cells than in stelar cells under waterlogged conditions. Interestingly, the MT gene was constitutively expressed in both cortical cells and stelar cells under aerobic conditions, but the MT mRNA levels were decreased specifically in cortical cells under waterlogged conditions. In addition, the use of diphenylene iodonium (DPI), an inhibitor of RBOH activity, reduced the aerenchyma formation in the root cortical cells treated under waterlogged conditions.

The last step of aerenchyma formation involves cell wall loosening and degradation, in which many enzymes are involved. Genes related to cell wall degradation were also selected from the microarray experiment. Among the up-regulated genes, genes encoding xyloglucan endo-transglycosylase (XET), cellulose (CEL) and polygalacturonase (PG). These genes are related to cell wall loosening and degradation. The tissue specificity analysis showed that, XET and CEL genes were specifically expressed in the root cortical cells under waterlogged conditions. However, the XET was up-regulated in both cortical cells and stelar cells. A gene encoding plasma membrane (PM) H+-ATPase as the cortical cell-specific up-regulated gene. It is proposed that extrusion of an intracellular H+ into the cell wall by PM H+-ATPase results in a decrease of apoplastic pH, which induces cell wall loosening, possibly mediated by low-pH activated XETs.

Many studies have suggested that the cytosolic calcium ion (Ca2+) functions as a second messenger for signaling pathways in response to oxygen deprivation. Ca2+ signaling may also be involved in aerenchyma formation in maize roots. In this study, several genes implicated in calcium signaling such as genes encoding calcineurin B-like protein (CBL), Ca2+-binding domain containing proteins and calmodulin-like protein (CML), were selected. The expressions of CBL and CML were observed in both cortical cells and stelar cells under waterlogged conditions, but the changes in expression were more pronounced in cortical cells than in stelar cells. However, the expression of the cyclic nucleotide-gated ion channel 2 (CNGC) gene was repressed under waterlogged conditions.

In conclusion, in this study, genes related to many types of molecular function (e.g. ROS generation or scavenging, cell wall modification, and Ca2+ signaling) were found as up-regulated or down-regulated in root cortical cells under waterlogged conditions, and their expression was likely to be regulated by ethylene. The data should provide a basis for an understanding of the molecular mechanism of inducible lysigenous aerenchyma formation in plants.

本研究は、トウモロコシの通気組織形成過程で特異的に発現する遺伝子の網羅的な解析を行うことで、通気組織形成の分子機構の一端を解明したものであり、次の3つの章から構成されている。

1.湛水条件下および好気条件下におけるトウモロコシ幼植物体の成長および通気組織形成パターンの解析

トウモロコシ幼植物体の地上部と根の生育および通気組織形成パターンの解析を行うため、3日間好気条件下で生育させた幼植物体を、さらに湛水条件下または好気条件下で24時間生育させた。好気条件と比較して湛水条件では根の生育に明らかな遅延がみられた。一方、地上部の生育に違いは見られなかった。24時間の湛水処理の後に、根の様々な部位で通気組織形成パターンの解析を行った結果、基部から1.5-2cmの部位で特に顕著な通気組織形成が観察されたことから、この部位を材料にして、遺伝子発現解析を行うことにした。

2.レーザーマイクロダイセクション法およびマイクロアレイ解析を用いた通気組織形成に関与する遺伝子の同定

植物ホルモンのエチレンがトウモロコシの通気組織形成に関与することが知られている。そこで、3日間好気条件下で生育させたトウモロコシの幼植物体を1ppmのエチレンで処理して、種子根における経時的な通気組織形成率の解析を行った。その結果、処理後6-12時間の間に通気組織形成が開始されることが明らかとなった。一方、好気条件下では24時間後まで通気組織形成は全く観察されなかった。湛水条件下では、処理後18-24時間の間に通気組織が形成されたが、エチレン受容阻害剤である1-methylcyclopropene (1-MCP;1ppm)で前処理した湛水条件下では24時間後まで通気組織は形成されなかった。これらの結果から、湛水処理による通気組織形成はエチレンを介して行われることが確認できた。

通気組織は根の皮層において形成されることから、好気条件と湛水条件、1-MCPの前処理後に湛水処理する条件と1-MCPの前処理をせずに湛水処理をする条件で12時間生育させた種子根の皮層を、レーザーマイクロダイセクション法によって単離して、マイクロアレイ解析を行った。その結果、42,034プローブのうち575遺伝子が2つの比較条件において共通して変動することが明らかとなった。それらのうち、239遺伝子は2条件で共通して発現上昇しており、336遺伝子は2条件で共通して発現減少していることが分かった。それらの遺伝子の中には、活性酸素種の発生・消去、細胞壁の弛緩・分解、Ca2+シグナリングに関与する遺伝子が含まれていた。

3.半定量RT-PCR法によるマイクロアレイ解析結果の検証および組織特異的な遺伝子発現解析

第2章で行ったマイクロアレイ解析の結果から、発現上昇する遺伝子の中に、活性酸素種の発生に関与するNADPH oxidase関連タンパク質〔Respiratory burst oxidase homolog (RBOH)〕をコードする遺伝子が含まれていた。一方、発現減少した遺伝子の中には、活性酸素種の消去に関与するmetallothionein (MT)をコードする遺伝子が含まれていた。さらに、これらの遺伝子の組織特異的な発現を解析するために湛水条件下または好気条件下で12時間生育させた種子根の皮層組織と中心柱をレーザーマイクロダイセクション法によって単離した。これらの組織を用いて、半定量RT-PCRを行った結果、RBOH遺伝子の発現は湛水条件下で誘導され、発現は中心柱と比較してわずかに皮層で高いことがわかった。一方、MT遺伝子は好気条件下において両組織で構成的に発現しているのに対して、湛水条件下では皮層組織で特異的に発現が抑制されることが明らかとなった。これらの結果から、通気組織形成過程においてはエチレンの下流のシグナルとして活性酸素種が重要な役割を担っていることが示唆された。そのことを確認するために、湛水条件下においてNADPH oxidaseの阻害剤であるdiphenyleneiodonium (DPI) 処理を行ったところ、通気組織形成が抑制された。

通気組織形成の最終的な段階では、細胞壁の弛緩・分解に関わる複数の酵素が関与することが知られている。本研究によって、発現上昇した遺伝子の中には、細胞壁の弛緩・分解に関与するxyloglucan endo-transglycosylase (XET)、cellulase (CEL)、polygalacturonase (PG)をコードするものが含まれていた。組織特異的な遺伝子発現解析の結果、CEL遺伝子は湛水条件下において皮層組織で特異的に誘導されたが、XET遺伝子は皮層組織と中心柱の両方で発現が誘導されることが明らかになった。また、Ca2+シグナリングに関わるcalcineurin B-like (CBL)、calmodulin-like protein (CML)などの遺伝子が発現上昇していることが明らかになった。一方、cyclic nucleotide-gated ion channel (CNGC)をコードする 遺伝子は湛水条件下において、皮層組織で特異的に抑制されていることが明らかとなった。

これらの結果より、本研究ではトウモロコシの種子根の通気組織形成過程において、活性酸素種の発生・消去、細胞壁の弛緩・分解、Ca2+シグナリングに関与する遺伝子などが、エチレンの制御下で皮層組織特異的に誘導または抑制されることを、世界に先駆けて発見することができた。これらの知見は、作物の耐湿性の付与に重要な通気組織の形成機構を解明するための基盤となることから、学術上価値が高いといえる。したがって、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があるものと認めた。