The process of development continues throughout the plant lifetime. Unlike animals in which the organs are all produced during embryogenesis, in plants new organs are produced from the meristems post-embryonically. The shoot apical meristem (SAM) gives rise to the overground tissues such as leaves and flowers while the root apical meristem (RAM) gives rise to the underground tissues such as the root system. In addition to the continuing organ formation at the periphery of SAM, meristems are also continuously generated. The newly generated meristem at the axil of leaf is called axillary meristem (AM). AMs produce a few leaves and become axillary buds. Axillary buds may continue to grow and form branches or become dormant and arrest their growth. The phytohormones, auxin and cytokinin, are thought to play major roles in the control of axillary bud growth. Moreover, strigolactone (SL) was recently discovered as a novel phytohormone which suppresses shoot branching.

In rice, axillary buds grow as tillers that bear the panicle. The tiller number is one of the most important traits for rice production. It has been demonstrated that the genetic framework of SL biosynthesis, signaling and function are well conserved in rice. Furthermore, several key genes in the control of tiller growth have been identified through genetic analysis and QTL analysis. However, in spite of these recent extensive progresses, our understanding of the mechanisms controlling tiller outgrowth is still unsatisfactory. In this study, I first performed detailed analysis of rice tiller bud development and transcriptome analysis of tiller buds. Then I analyzed the interaction between SPL14, a major regulator of tiller growth, and the SL pathway.

Observation of tiller bud development

The pattern of rice tiller growth is determined by both endogenous and environmental factors. Some of tiller buds become dormant and stay as tiller buds until the condition becomes suitable for their growth. To understand the mechanisms that confer the dormancy to the tiller buds, first, I set up the growth condition in which tiller growth can be reproducibly observed. The second bud (the bud at the axil of the second leaf) was chosen for the analysis. The hydroponic culture system was used and the culture condition in which the second bud becomes dormant was established. In this culture system, the second bud of d10-2, a SL deficient mutant, continues to grow without entering into the dormant state. To determine the timing when the dormancy takes place, bud growth pattern was compared between WT and d10-2. In this study, new counting system was used. When the third leaf fully expanded is 3.0L stage which is equals to P (Plastochron) 5.5 in plastochron counting system, and when the fourth leaf half expanded is 3.5L equals to P6.0. At 3.5L stage, the growth of the second bud almost stopped in WT. This indicated that the transition of the bud phase from active to dormant takes place by 3.5L stage. The growth phase of the tiller buds changed between 3.0L and 3.5L stages. RNA in situ hybridization of a cell cycle marker gene Histone H4 showed that, in the second bud, cell division almost stopped by 3.5L stage in both meristem and leaf primordia of WT. The arrest of cell cycle was specifically observed in the tiller bud and Histone H4 expression level was not significantly changed in the SAM. The expression pattern of OSH1, a marker of the meristematic cells, was also analyzed. Although OSH1 signal was detected in the dormant buds at 3.5L stage, the signal was weaker compared to that in active buds and gradually disappeared. This indicated that the dormant bud probably remains the meristematic identity for a period. In conclusion, the second bud of WT at 3.5L stage is dormant in my experiment condition which is dependent on the endogenous SL.

Transcriptome analysis of the phase shift to dormant state in tiller buds

In order to find out the factors that trigger the dormancy in tiller buds, microarray analysis was carried out. Changes of gene expression profiles were compared between active and dormant buds of WT plants sampled at 9 day (3.0L) and 11 day (3.5L), respectively. The buds were also sampled at 10 day and analyzed as transition stage buds. Buds were sampled from d10-2 plants in which buds are maintained at the active state. To analyze gene expression profiles specifically related to bud dormancy, tiller buds including the axillary meristem and youngest two leaves were sampled using laser capture microdissection technique and used for RNA isolation. The overall gene expression levels were higher in dormant buds compared to active buds. In total, 1,718 genes were up-regulated (fold change more than 2) while 829 genes were down-regulated (fold change more than 2) in dormant buds. This implied that an active mechanism probably operates to cause the phase transition from active to dormant state in the tiller buds. An enrichment of ribosomal protein genes was a prominent feature in down-regulated genes. All four homologs of dormancy-associated genes were up-regulated in dormant buds, supporting the relevance of our experimental system to analyze bud dormancy. Expression levels of cell cycle related genes were also changed. Expression of EL2 and EL2-like, putative plant specific cell cycle inhibitors, were increased in dormant buds. It was well known that Abscisic Acid (ABA) is involved in seed dormancy and seasonal bud dormancy of perennial trees. In my microarray data, extensive increase of ABA inducible genes was observed. Moreover, genes involved in ABA biosynthesis and catalysis were also increased. In the contrast to the extensive changes of gene expression levels in WT bud, much less changes were observed in d10-2. All these results indicate that ABA may be involved in the control of bud dormancy and the changes are dependent on SL.

Genetic analysis of SPL14 and SL

SPL14, a member of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes, is known to suppress outgrowth of tiller buds and to regulate panicle development in rice. The mechanism of SPL14 on tiller outgrowth is unclear. First, the function site of SPL14 was examined. I showed that SPL14 mRNA accumulated in leaf primordia during the vegetative phase and in the primordia of bracts, or modified leaves, in the panicles, but not in the meristems. Next, genetic interaction between SPL14 and SL was analyzed. SPL14 is a target of miR156 and accumulation of SPL14 transcripts is negatively regulated by miR156. The enhancement of the expression level of SPL14 by the introduction of the mSPL14 gene, in which the miR156 cleavage site is mutated, resulted in a decrease in tiller number in both WT and in d10-2, a SL-deficient mutant. Expression of miR156 by a constitutive CaMV 35S promoter dramatically increased tiller number. The enhanced tiller growth was suppressed by an application of SL. There analysis suggested that SPL14 and SL function in parallel pathways to suppress tiller growth. SL exuded from roots trigger germination of root parasitic plants which causes severe damage to crop productivity. SL-deficient mutants, however, exhibit an excess branching phenotype which is usually undesirable for productivity. Introduction of the mSPL14 gene in the d10-2 mutant suppressed over-branching defects in d10-2 in an expression dependent manner. This indicated that SPL14 can be used to manipulate the branching patterns of SL-deficient mutants. We also confirmed that this strategy is applicable to Arabidopsis. A greater understanding of the SPL14 and SL pathways and their interactions may help in the production of root parasite-resistant crops.

In conclusion, the analysis of rice tiller bud development discovered the time when the tiller bud stops growing and becomes dormant. The transcriptome analysis of tiller bud at active and dormant phase revealed the basic information of buds and suggested that cell cycle genes and ABA were involved in the control of bud dormancy. The investigation of SPL14 and SL pathway showed that the regulation of plastochron is important for SPL14 on controlling tiller outgrowth which is independent of SL.

植物は成長段階や環境条件に応じてその形態や大きさを変化させる。特に、分枝パターンの調節は植物が環境と調和して効率よく成育するために非常に重要である。分枝は葉の腋につくられる腋芽(えきが)が成長することにより形成される。腋芽はつねに伸長するわけではなく、形成された後に成長を止め(休眠)、伸長のための条件が整うと休眠が解除され分枝として伸長する。イネでは、腋芽は分げつとして成長しその先端に穂が形成されるため、その休眠の制御は収量決定の重要な要因である。最近、ストリゴラクトンが腋芽を伸長させる植物ホルモンであることが明らかになったが、依然、腋芽休眠現象の分子レベルでの理解は進んでいない。本論文では、腋芽の休眠を制御する機構の理解を目的として、イネ分げつ芽の休眠開始機構を詳細に解析した。また、イネ分げつ伸長に関わるSPL14 遺伝子の育種への利用可能性が検討された。

第1章 緒言

第2章

本章ではイネ分げつ芽の休眠を解析するための実験系を確立した。イネでは、植物体の成長に伴い規則正しく葉が分化し、その付け根に腋芽が形成される。まず、育成条件によりイネ(品種:日本晴)第2葉(2番目の葉)の腋芽が伸長するか休眠するかが決定されることを確認し、再現性良く腋芽休眠開始を観察できる水耕栽培条件を確立した。腋芽の伸長抑制に必要な植物ホルモンであるストリゴラクトンを合成しないdwarf10(d10) 変異体との比較から、この実験系では植物体の成長段階が3葉齢から3.5葉齢に至る二日間に第2葉の腋芽が休眠することを示した。さらに、細胞周期遺伝子の発現解析を行い、腋芽が休眠を開始する際には腋芽での局所的な細胞分裂の停止が起こることを見出した。また、植物の幹細胞としてはたらくメリステムでは、休眠にともなってメリステム活性が徐々に低下することを示した。細胞分裂の停止や腋芽の伸長抑制はストリゴラクトン依存的であり、d10変異体にストリゴラクトンを与えると濃度依存的に腋芽が休眠することを示した。

第3章

腋芽休眠に伴う遺伝子発現変化を解析するために、レーザーマイクロダイセクション法を利用して野生型イネ(日本晴)の第2葉腋芽をサンプリングし、マイクロアレイにより休眠開始時の遺伝子発現変化を解析した。比較のためにd10変異体でも第2葉腋芽の遺伝子発現を解析した。この解析により腋芽休眠時に発現レベルが変化する遺伝子群が単離された。まず、休眠腋芽で発現が上昇する遺伝子として報告されているDormancy Related (DR)遺伝子の4つのイネオーソログの発現が上昇していたことから実験系の妥当性が示された。本解析から得られた重要な結果は、腋芽の休眠開始においては多数の遺伝子の発現が上昇するということであった。腋芽休眠にともない、1,877遺伝子の発現が2倍以上上昇したのに対し、発現が2分の一以下に低下した遺伝子は995であった。この結果は、植物は多数の遺伝子を発現させて積極的に腋芽の成長を停止させていることを意味する。

最大の発現上昇を示した遺伝子は機能未知のアブシジン酸(ABA)誘導性遺伝子であり、また多数の既知のABA誘導性遺伝子の発現が上昇した。この結果から、腋芽休眠とABAの関係が示唆された。また、細胞周期を制御する遺伝子群のうち、細胞周期を促進するサイクリンAおよびサイクリンB遺伝子の低下が認められた。また、植物特異的な細胞周期抑制遺伝子(EL2)の発現が休眠腋芽で顕著に上昇することが特徴的であった。

第4章

ストリゴラクトンは腋芽を休眠させる植物ホルモンである。また、根から分泌され菌根菌との共生を促進すると同時に根寄生植物の発芽を促進する。したがって、ストリゴラクトンを合成しないd10変異体は根寄生植物に耐性であるが、分げつが過剰に成長してしまうために栽培には適さない。この問題を克服するために、既知の腋芽伸長抑制遺伝子(SQUAMOSA PROMOTER BINDING PROTEIN LIKE14 (SPL14))の利用を試みた。d10変異体でSPL14遺伝子の発現を上昇させると、ストリゴラクトンを合成しないにもかかわらず、分げつの成長が正常に回復した。SPL14遺伝子はストリゴラクトンとは独立に作用することが示された。また、イネSPL14遺伝子がシロイヌナズナのストリゴラクトン合成・信号伝達変異体においても腋芽の過剰伸長を抑制したことから、同様のストラテジーがシロイヌナズナにも応用できることが確認された。

以上、本論文では腋芽の休眠という植物の成長にとって重要な現象を分子レベルで理解するための基礎的知見が得られた。腋芽休眠を時間的・空間的にピンポイントで解析し、休眠にともなう遺伝子発現の変化をゲノムワイドに捉えたことは有意義な成果である。また、得られた知見は腋芽休眠という植物に普遍的な現象の理解だけにとどまらず、作物栽培や育種への応用の可能性を秘めている。以上、本研究で得られた知見は、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。