In this thesis I describe analyses on plant response and tolerance to nutrient deficiency in model plant Arabidopsis thaliana.

There are seventeen essential elements for plant growth and reproduction (Cakmak and Romheld 1997). Among them, nitrogen, phosphorus, and potassium are often added to fields as fertilizers to improve crop production. This means these major nutrients are deficient in many soils. Some other nutrients (minor nutrients) are also deficient in soils, and cause agricultural problems around the world. Diverse methods have been adopted by researchers to reveal genetic mechanisms for response and tolerance to nutrient deficiency. Straight-forward screening of deficiency-sensitive loss-of-function mutants was only successful in restricted numbers of studies, including identification of LKS1 which regulates a potassium transporter AKT1 (Xu et al. 2006). There are also reports on identification of nutrient transporters with the use of toxic analogs, for example identification of sulfate transporter SULTR1;2 from selenate-tolerant mutants (Shibagaki et al. 2002) and identification of silicon transporters OsLsi1 and OsLsi2 from germanium-tolerant mutants (Ma et al. 2002, Ma et al. 2006, Ma et al. 2007).

Induction of expression by nutrient deficiency is also a cue to identify genes. OsPTF1, a transcription factor which improves rice growth under phosphorus deficiency, was identified from its induction by phosphorus deficiency (Yi et al. 2005) and a boron transporter NIP5;1 was identified from its induction by boron deficiency (Takano et al. 2006). Several other methods were taken to identify various genes involved in the tolerance to nutrient deficiency (for only a small part of examples, Hirsch et al. 1998, Hamburger et al. 2002).

In this study, I aimed to reveal novel genetic mechanisms that improve plant tolerance to nutrient deficiency. As described above, there have been several strategies to take for this goal, and I took the same ways (chapters 1,3,4) or I also tried a novel method (chapters 5,6) in this study. In chapter 2, I established a novel high-throughput method to accelerate genetic studies. Although these extensive trials did not identify genes that improve plant tolerance to nutrient deficiency, I identified several novel aspects of plant response to nutrient deficiency. Most importantly, recent results indicate novel methods to improve plant tolerance to nutrient deficiency through modification of functionally characterized genes.

Chapter 1. The BIG gene is involved in regulation of sulfur deficiency-responsive genes in Arabidopsis thaliana

asr1 is one of the low-sulfur response mutants isolated by Dr. Naoko Ohkama-Ohtsu. Expression of green fluorescence protein gene driven by a low-sulfur responsive promoter is upregulated in this mutant. After collaboratory map-based cloning, nonsense mutations were identified in the BIG gene locus in both two alleles. Low-sulfur response was also upregulated in other big alleles. To know the factor which upregulate low-sulfur response in big mutants, indole-3-acetic acid and an auxin transport inhibitor was applied to plants, because disturbed auxin transport is the most important effect know to be caused by big mutation. These two chemicals induced low-sulfur responses in Arabidopsis, although in some different pattern from those observed in big mutants. Thus we concluded that the BIG gene affects low-sulfur response independently of auxin metabolism.

Chapter 2. A protocol for rapid DNA extraction from Arabidopsis thaliana for PCR analysis

DNA extraction protocols from Arabidopsis required several steps, such as boiling, ethanol precipitation, or drying in vacuum. A number of simplified and rapid protocols of DNA extraction for Arabidopsis have been reported, however, they still required several steps. Here I established a novel one-step DNA extraction protocol for uses in polymerase chain reaction (PCR). Based on several rapid protocols, I tested several conditions. Among them, a buffer which is a dilution of a former extraction buffer (Edwards et al. 1991) successfully extracted DNA in one-step. The DNA solutions were successfully analyzed for detection of polymorphisms between Arabidopsis accessions or detection of T-DNA insertion (performed by Ms. Yoko Ide) by PCR. Other rapid one-step protocols are also reported by other research group (Berendzen et al. 2005). These protocols are suitable for saving time, labor, and cost of DNA extraction from Arabidopsis.

Chapter 3. Identification of novel Arabidopsis thaliana genes which are induced by high levels of boron

In tobacco BY-2 cultured cells, several genes induced by boron deficiency are reported (Kobayashi et al. 2004). In Arabidopsis, NIP5;1 was the only gene which was known to be induced by boron deficiency. NIP5;1 is a boron channel which rescues plant growth under boron deficiency (Takano et al. 2006). To know the transcriptome-level regulation of gene expressions by boron deficiency and boron toxicity, transcriptome analysis was performed in Arabidopsis. Among ~12,000 genes detected in this analysis, NIP5;1 was the only gene whose expression was induced only by boron deficiency more than 2.5-fold, indicating specific mechanism for regulation of NIP5;1 gene expression by boron deficiency. In this analysis several genes whose expressions are induced only by boron toxicity were also identified. This is the first identification of high-boron induced genes.

Chapter 4. Regulation of gene expression by boron deficiency around root tip of Arabidopsis thaliana and involvement of WRKY6 in regulation

To know the functions of high/low-boron responsive genes identified in chapter 3, mutants which possess T-DNA insertions in the exons of the induced genes were selected. These mutants were analyzed for tolerance to high/low-boron. Mutant designated as wrky6-3 showed reproducible but not stable phenotype. Root elongation of wrky6-3 was sometimes worse than wild-type under boron deficiency. Promoter activity of the WRKY6 gene was constantly induced near the root tip under boron deficiency. WRKY6 is a transcription factor. To know the effect of WRKY6 on gene expressions under boron deficiency, another transcriptome analysis was performed around root tip. Many genes were induced by boron deficiency around root tip after a long-term boron deficiency. Inductions of some of these genes were inhibited in wrky6-3 mutant, showing involvement of WRKY6 in regulation of gene expressions by boron deficiency.

Chapter 5. Screening of Arabidopsis thaliana gain-of-function mutants under nutrient deficiency

Screening of loss-of-function mutants like ethylmethanesulfonate-mutagenized lines has been applied to screenings for genes functioning in tolerance to nutrient deficiency, although only restricted numbers of successful screenings are found (ex. Xu et al. 2006). This will be partially because naturally smaller mutants also appear in the screening and it is hard to distinguish sensitive individuals to nutrient deficiency from naturally smaller mutants. We should also be aware that a gene which functions for tolerance to nutrient deficiency does not necessarily improve growth when ectopically overexpressed. Screening of gain-of-function mutants may be an effective alternative. In this chapter, screening of gain-of-function mutants was performed under nutrient deficiency for the first time. After screening ~450,000 plants, although, no obviously tolerant line was identified. Because control genes such as LKS1 (Xu et al. 2006) and BOR1 (Miwa et al. 2006), which improve tolerance to potassium deficiency and boron deficiency, were also not recovered in the screening, the number of plants was not enough in the screening. Developments of high-throughput screening conditions are waited for for completion of the screening. In FOX lines, position effect will reduce screening efficiency. Introduction of sea urchin insulator sequence upstream of 35S promoter in FOX lines may improve screening. On the other hand, several larger lines in size were recovered in the screening. Because properties of cell ploidy were not largely different between mutants and wild-type, a novel mechanism will improve sizes of these mutants.

Chapter 6. Differential regulation of root architecture in autotetraploid Arabidopsis thaliana under boron deficiency

In the screening performed in chapter 5, three long-root mutants under boron deficiency were isolated. Morphology of one of these mutants, designated as A152B, was similar to tetraploid, such as bigger seeds, bigger flowers, and bigger pollens. To confirm ploidy, cell ploidy was measured in wild-type and A152B. The peak for nuclei containing two sets of chromosomes existent in wild-type was not observed in A152B. Thus A152B was autotetraploid. Root elongation of other autotetraploid lines obtained from stock center was also improved under boron deficiency. Fresh weights of both root and shoot were heavier in tetraploid or triploid under normal condition. Because proportion of fresh weights between diploid and polyploids was not largely altered under boron deficiency, polyploids are not tolerant to boron deficiency but are differentially regulated in their root morphology under boron deficiency. Elongation of root cells is inhibited under boron deficiency. This inhibition was milder in tetraploid, which causes improved root elongation under boron deficiency.

Discussion

In this thesis, several responses of Arabidopsis to nutrient deficiencies and toxicity were revealed. Although the extreme goal of the study was to improve plant tolerance to nutrient stresses, no novel strategy to attach tolerance to nutrient stresses was identified. Screening of gain-of-function mutants, performed in chapters 5 and 6, was a novel method and I expected to isolate various genes. The failure of the screening indicated incompleteness of the screening and necessity to improve screening systems.

Several nutrients are deficient in some or many crop fields. Fertilizers are added to these fields to support crop production. Nitrogen and phosphorus fertilization can cause enrichment of these elements in environment and each nutrient can threat species richness (Stevens et al. 2004, Wassen et al. 2005). Another point to stress is that some estimations predict depletion of inexpensive phosphorus ores for fertilizer in the world by 2050 (Vance et al. 2003). Improvement of crop uptake and use efficiency of nitrogen and phosphorus is an important approach to settle these severe situations.

The number of endogenous genes which improve plant tolerance to nutrient deficiency is restricted. This may indicate that Arabidopsis and other plant species evolved nearly fully to adapt to nutrient deficiencies, and not allowing further tolerance attached by modification of endogenous genes. A recent study in our laboratory, on the other hand, indicates difference between ectopic overexpression under the control of 35S promoter and activation through combination of 35S enhancer and native promoter of an endogenous gene. If this is true to that gene, the same phenomenon could be observed in other genes. My recent trial also supported this phenomenon. Although further confirmations are necessary, these results indicate that tolerance to nutrient deficiency is attached to plants only by activating functionally identified endogenous genes. This may be an important trial for sustainable agriculture.

After identification of genes which improve plant tolerance to nutrient deficiency, can we modify crops without transgenes? We can find an example in which bread wheat was reverse-genetically improved without transgenes by TILLING (Slade et al. 2005). Although mutation is randomly inserted in TILLING and most mutations are restricted to C to T or G to A transitions consistent with guanine alkylation (Colbert et al. 2001, Slade et al. 2005), sequential TILLING and backcrossing may enable modification of promoter sequences or modification of codon usage through accumulation of silent mutations in favor of major codons, for activation of gene expressions. Or alternatively, some transposons enhance expressions of nearby genes or may shut down the spreading of heterochromatins along chromosome from original target sites of transcriptional gene silencing onto the target genes. For application of basic knowledge in plant genetics to improvement of crop tolerance to nutrient deficiency, I find several very challenging but not impossible studies, which should be performed to save our future.

本論文はモデル植物であるシロイヌナズナにおける栄養欠乏への応答や耐性について研究したものである。序章に引き続く6章より成る。

序章では以下の点を中心に植物の栄養研究の現状を述べている。植物の生育や繁殖に必要な必須元素が17個知られている(Cakmak and Romheld 1997)。これらのうち、窒素、リン酸、カリウムは三大栄養素と呼ばれ、広く農耕地に散布され農業生産の向上に寄与している。この三つ以外の微量元素についても、土壌で欠乏し農業上の問題を引き起こす例が世界中で知られている。このような栄養欠乏に対する応答や耐性に関する遺伝的な機構を明らかにするために、研究者はこれまで様々な手法を用いてきた。機能喪失変異株をスクリーニングするという手法は、カリウムのチャネルを制御するLKS1の同定等、いくつかの成功例が報告されている(Xu et al.2006)。これ以外にも、毒性アナログ物質に耐性の変異株の解析や栄養欠乏による発現誘導、その他の手法により、多くの栄養欠乏耐性に関わる遺伝子が同定されている。

第一章ではBIG遺伝子はシロイヌナズナの硫黄欠乏応答性遺伝子の制御に関わっていることを示している。asr1は大鎌直子博士により単離された硫黄欠乏応答変異株の一つである。この変異株では、硫黄欠乏応答性遺伝子の発現が強まっている。共同で行ったマッピングにより、この変異株では1野0遺伝子にナンセンス変異が挿入されていることが分かった。また、他のbig変異株でも硫黄欠乏応答が誘導されていた。big変異株ではオーキシン輸送が阻害されていることから、オーキシンやオーキシン輸送阻害剤を植物に添加してみると、これらの処理により硫黄欠乏応答が誘導された。ただ一方で、この誘導パターンはbig変異株のそれとは異なっており、BIG遺伝子はオーキシンを介さずに硫黄欠乏応答に影響していると考えられた。

第二章ではPCRのためのシロイヌナズナからの迅速DNA抽出法の開発について述べている。シロイヌナズナからのDNA抽出は従来、煮沸、エタノール沈殿、減圧下での乾燥といったいくつかのステップを必要としていた。そこで私は迅速な手法を試み、PCRに適したDNAをワンステップで抽出する方法を開発した。具体的には、既知の方法に基づき幾つかの簡便法を試みた。その中で、ある抽出バッファー(Edwards et al.1991)を希釈した溶液を用いることによりワンステップでDNAを抽出することに成功した。このDNA溶液を用いることにより、シロイヌナズナのアクセッション間の多型を判別したりTDNAの有無(井出曜子さんのデータ)を確認することができた。ここで開発したようなワンステップ法を用いることにより、シロイヌナズナからのDNA抽出を行う際の手間、時間、コストを削減することができる。

第三章ではホウ素過剰により発現が誘導される新たなシロイヌナズナ遺伝子の同定について報告している。シロイヌナズナでホウ素欠乏により発現が誘導される遺伝子として、ホウ素チャネルであるNIP5;1が知られていた(Takanoet al.2006)。ホウ素欠乏やホウ素過剰により発現が誘導される新たな遺伝子を同定するため、トランスクリプトーム解析を行った。その結果、ホウ素欠乏特異的に強く発現誘導を受ける遺伝子としてはNIP5;1だけが検出された。一方、ホウ素過剰により発現が誘導される遺伝子が、初めて同定された。

第四章ではシロイヌナズナの根の先端周辺でのホウ素欠乏による遺伝子発現調節とWRKY6の関与について述べている。第三章で同定された、ホウ素欠乏や過剰により発現誘導を受ける遺伝子のいくつかについて、その機能を調べるためにTDNA挿入変異株を入手した。このうち、WRKY6という転写因子にTDNA挿入を持つwrky6-3変異株の生育がホウ素欠乏条件下で野生型株と異なっていた。違いが見られないこともあるものの、差が顕著な場合にはホウ素欠乏条件で栽培した変異株の根は野生型株の3分の2程度にまで短かった。また、WRKY6遺伝子のプロモーター活性はホウ素欠乏により根の先端付近で誘導され、マイクロアレイを行うとWRKY6-3変異株でホウ素欠乏による誘導が抑制される遺伝子が見られた。

第五章では栄養欠乏条件下でのシロイヌナズナ機能獲得変異株のスクリーニングを行っている。栄養欠乏条件下で機能喪失変異株をスクリーニングするというアプローチは遺伝学として一般的な手法であるが、成功例は限られている。また、機能喪失で表現型を示す遺伝子は必ずしも機能獲得で表現型を示すものではない。そこで、機能獲得変異株を栄養欠乏条件でスクリーニングすることにより栄養欠乏耐性を植物に与える遺伝子を効率よく直接的に同定できるのではないかと考え、スクリーニングを行った。約45万個体を栄養欠乏条件等でスクリーニングしたが、残念ながら明らかに欠乏耐性である変異株は得られなかった。一方で、サイズが通常条件で栽培した際にも大きい変異株が得られたことを述べている。

第六章では自己四倍体シロイヌナズナにおける根の構造のホウ素欠乏条件下での異なる制御について述べている。第五章ではホウ素欠乏条件で根が長い3つのアクチベーションタグラインも単離されていたが。このうちA152Bと名付けたラインは形態的に四倍体に似ていた。cell ploidyを測定すると、確かにこのラインは四倍体だった。A152Bはホウ素欠乏条件下でのみ二倍体よりも明らかに根が長い。ストックセンターから入手した他の四倍体も同様の生育を示したことから、これが自己四倍体の一般的な表現型であることが分かった。新鮮重の比率は通常条件とホウ素欠乏条件で変わらないことから、このフェノタイプはホウ素欠乏に対する耐性というよりは、ホウ素欠乏に対する応答が異なっていると言える。四倍体で環境ストレス応答が変化しているということはこれまで知られておらず、四倍体の新たなタイプの性質が本研究により明らかとなった。

以上、本論文は、シロイヌナズナの栄養欠乏や過剰に対する応答に関して新たな現象をいくつか明らかにしたものであり、植物栄養の分野において極めて高い貢献をしている。

よって、審査委員一同は、本論文を博士論文として高く価値あるものと認めた。