The molecular mechanisms of boron (B) toxicity are not well-understood. In order to obtain insights into the molecular mechanisms of B toxicity, seven Arabidopsis thaliana mutants hypersensitive to excess B (heb) were studied. Through the analysis of the mutants, I identified six genes involved in B toxicity tolerance. These genes represent the first identification of genes essential for B toxicity-tolerance in plants.

Introduction

Boron (B) is an essential element for plants. It can also become toxic when it exists in soils at excessive levels. Limitation of crop yield and quality caused by B toxicity is an agricultural problem in the world especially in semi arid areas.

To understand B toxicity mechanisms and breed excess-B tolerant crops, isolation of genes involved in B toxicity and/or tolerance has been attempted. Recently, overexpression of B transport molecules BOR4 and TIP5;1 were revealed to improve excess-B tolerance in plants, although the biological functions of these molecules in B toxicity have remained unclear. These studies established that regulations of molecules function in efflux and uptake of B are major mechanisms for excess-B tolerances in plants. On the other hand, at the molecular level, mechanisms of excess-B toxicity are still unknown. Other than B transport molecules, several A. thaliana proteins involved in transcription, RNA process and anti-oxidative system are shown to provide B tolerance to yeast. However, their functions in B toxicity-tolerance are not revealed yet. Moreover, it has not been elucidated whether these genes are essential for B toxicity-tolerance in plants. Isolation and identification of novel plant genes involved in B toxicity and/or tolerance is expected to provide us new insights into molecular mechanisms of B toxicity in plants.

For this purpose, I focused on genetic approach using EMS mutagenized Arabidopsis thaliana (ectype Col-0). I studied seven recessive mutants, hypersensitive to excess B (heb). The heb mutants showed extremely shorter relative root length than the wild type under the toxic B condition (3mM boric acid), although they showed slightly reduced root elongation under the control condition (0.03mM boric acid), indicating their hypersensitivity to excess B. In the present thesis, I first identified the genes that are essential for B toxicity-tolerance in plants using the heb mutants and characterized their functions in B toxicity-tolerance. Through the analyses, I established new aspects of two protein complexes, condensin II and 26S proteasome.

Chapter 1 Condensin II alleviates DNA damage and is essential for excess boron tolerance in Arabidopsis thaliana

First I investigated the mineral specificity of the short-root phenotype of heb1-1 and heb2-1. The root growth of heb1-1 and heb2-1 were not sensitive to B deficiency, arsenite toxicity and salinity stress, indicating that short-root phenotype of heb1-1 and heb2-1 are specific to excess B among mineral stresses tested.

Genetic mapping and sequence analysis revealed that heb1-1 carried two mutations in At1g64960 which encodes chromosomal associated protein-G2 (CAP-G2) and that heb2-1 carried a mutation in At3g16730 encoding CAP-H2. Introduction of GFP-fused CAP-G2 and CAP-H2 into the respective heb mutants complemented their excess-B dependent phenotype, confirming that these are responsible genes of the heb mutants. Both proteins are subunits of chromosomal protein complex condensin II, suggesting that the function of condensin II complex is crucial for excess B tolerance in A. thaliana.

Condensin II is composed of two core subunits CAP-C and CAP-E and three regulatory subunits HEB1/CAP-G2, HEB2/CAP-H2 and CAP-D3. In human cells, condensin II is well known to have a role in mitotic chromosomal condensation in concert with another type of condensin, condensin I. In human cells, in addition to the mitotic function, condensins are known to be involved in DNA damage repair during interphase. To investigate whether A. thaliana condensin II is involved in DNA damage response as is the case in animal cells, I examined the sensitivity of HEB1/CAP-G2 and HEB2/CAP-H2 mutants to reagents/conditions that induce DNA double strand breaks (DSBs). The root growth of both heb1-1 and heb2-1 were sensitive to DSBs-inducible reagents compared to the wild type, suggesting the involvement of condensin II in DNA damage repair and/or in resistance to genotoxicity.

To examine whether excess B causes DNA damage, I investigated the expression of DSBs-inducible genes and the levels of DSBs in the root tip cells treated with excess B. RT-PCR revealed that expressions of DSBs-inducible genes such as BRCA1 and PARP1 were up-regulated by excess B treatment in both the wild type and the mutants. Transcripts of these genes were higher in the heb mutants than in the wild type both under the control and the excess B conditions. Comet assay revealed that the heb mutants highly accumulated DSBs compared to the wild type under the control and the excess B conditions. The levels of DSBs in both the wild type and the heb mutants were elevated by the excess B treatment. Taken together, these results demonstrate that excess B causes DSBs in root tip cells and A. thaliana condensin II has a role in the alleviation of DNA damage.

In conclusion, I demonstrated that involvement of DSBs in B toxicity and a novel function of plant condensin II in repairing damaged DNA and/or protecting genome from genotoxic stresses especially under the excess B condition.

Chapter 2 Involvement of 26S proteasome in excess B tolerance in Arabidopsis thaliana-Identification of possible targets involved in excess B tolerance-

Genetic mapping and sequence analysis revealed that heb3 carries a mutation in At5g05780 which encodes regulatory particle non-ATPase 8a (RPN8a) and heb6 in At3g05530 which encodes regulatory particle triple-A-ATPase 5a (RPT5a). heb7 had a mutation in RPT5a at a different site from heb6. These mutants were not much sensitive to B deficiency, cadmium, arsenite and sodium chloride toxicity as compared to excess B, indicating the specificity of the mutants to excess B tolerance. To avoid confusion, I renamed heb3, heb6 and heb7 as rpn8a-2, rpt5a-5 and rpt5a-6, respectively.

RPN8a and RPT5a are subunits of 19S regulatory particle (RP) of 26S proteasome (26SP), a large proteolytic device. RP functions in recognition and unfolding of target proteins which are mostly modified by ubiquitin (Ub). In A. thaliana, most of the RP subunits were encoded by two genes, suggesting a diverse subunit combination of 26SP is present, which may expand the target specificity and functions. Among T-DNA inserted RP mutants I examined, rpn2a and rpt2a mutants were also hypersensitive to excess B, but rpn2b, rpn8b, rpt2b and rpt5b mutants were not. This suggests the existence of specific combination of RP subunits and specific targets in response to B toxicity.

I elucidated whether the total Ub-dependent proteolysis activity is reduced in the rpt5a mutants. The rpt5a mutants were sensitive to treatment with amino acid analogue which induces accumulation of misfolded proteins. Misfolded proteins are known to be degraded through the Ub-26SP pathway. This suggests the reduced total Ub-dependent activity in the rpt5a mutants and that excess B may cause protein misfolding. On the other hand, the levels of accumulated poly-ubiquitinated proteins in the rpt5a mutants were not higher than those in the wild type under the normal B condition. This indicates that the accumulation of poly-ubiquitinated proteins does not reflect the reduced total Ub-dependent activity in the rpt5a mutants. Interestingly, the accumulations of poly-ubiquitinated proteins were increased by excess B in the rpt5a mutants, but not in the wild type. Taken together, these data suggest that RPT5a contained 26SP is involved in the degradation of those proteins induced by excess B.

To investigate whether the subunit specific poly-ubiquitinated proteins in response to excess B are present, I conducted proteome analysis of poly-ubiquitinated proteins. Poly-ubiquitinated proteins were purified from the root extracts of the wild type and rpt5a-6 and were analyzed by isobaric tag for relative and absolute quantification (iTRAQ) LC-MS/MS. As a result, 30 of 57 identified proteins were relatively quantified. Accumulations of 21 of 30 proteins were higher in rpt5a-6 than in the wild type irrespective of B condition and were elevated by excess B treatment, suggesting that those proteins are degraded through a pathway that requires RPT5a. Some of the identified proteins were known to associate to stress response and cell morphogenesis. One possibility is that these proteins undegraded are cause of excess B sensitivity.

As another approach to elucidate molecular mechanisms of RPT5a involvement in excess B tolerance, several revertants carries rpt5a-6 mutation and can elongate roots under the excess B condition were isolated. The revertants are expected to provide molecular information on the function of RPT5a in tolerance to excess B.

In conclusion, in this chapter, I demonstrated the requirements of RPN2a, RPN8a, RPT2a and RPT5a for B toxicity-tolerance. I propose that among a variety of compositions of 26SP, those containing RPN2a, RPN8a, RPT2a and/or RPT5a are crucial for excess B tolerance. These sets of 26SP may have essential function in B toxicity-tolerance through Ub-dependent proteolysis of certain proteins with negative effects on root growth.

Chapter 3 Arabidopsis thaliana 26S proteasome subunits RPT2a and RPT5a are crucial for Zinc deficiency-tolerance

Through the analysis of nutritional response of RP mutants, I found that the shoot growth of rpt2a and rpt5a mutants were sensitive to zinc (Zn) deficiency.

I first speculated that the rpt mutants are defective in the regulation of Zn uptake. However, in the rpt mutants, shoot Zn contents were similar to that of the wild type. On the other hand, transcripts of Zn deficiency-inducible genes, ZIP4 and ZIP9 were highly accumulated in the rpt mutants, suggesting the possibility that the rpt mutants are suffering from various Zn deficiency symptoms although Zn levels are not reduced.

Indeed, lipid peroxidation levels, known to be increased under Zn deficiency, were higher in the rpt mutants than in the wild type, suggesting that ROS accumulation in the rpt mutants are higher than in the wild type.

It has been known that up-regulation of 26SP subunit genes reflects the decrease in Ub-dependent 26SP activity in plants. Zn deficiency induced expression of both RPT2a and RPT5a genes, and the extents of induction of these genes were much higher in the rpt mutants, suggesting the reduced activities of Ub-dependent proteolysis under Zn deficiency, especially in the rpt mutants. Indeed, poly-ubiquitinated proteins were accumulated upon exposure to Zn deficiency, especially in the rpt mutants.

Overall, my analysis established that RPT2a and RPT5a are involved in Zn deficiency response, possibly through alleviation of oxidative stresses and/or processing of poly-ubiquitinated proteins.

Conclusion

Through the characterization of heb mutants, I identified six genes required for B toxicity-tolerance in plants and established novel aspects and mechanisms of B toxicity at the molecular level. In addition, the present thesis also provides novel aspects of condensin II and 26S proteasome function in nutritional responses.

ホウ素は植物に必須な微量栄養元素であるが、植物体内で過剰に存在すると毒性を示す。ホウ素過剰によって、細胞分裂や伸長の阻害、炭素・窒素固定能の低下、活性酸素種の発生に伴う酸化ストレスなどが起こり、結果として生育が阻害される。しかしながら、ホウ素過剰がもたらすこれらの負の現象がどのようにして起こり、そのプロセスにどのような遺伝子が関わっているのかは不明である。本論文は、根の生育を指標にして単離されていたホウ素過剰超感受性シロイヌナズナ変異株の解析を通じて、ホウ素毒性の分子機構を解明することを目的として研究を行ったものであり、序論と3章よりなる。

第一章では、変異株の原因遺伝子の単離と解析を行った結果、コンデンシンIIと呼ばれるタンパク質複合体のCAP-H2及びCAP-G2の2つのサブユニットをコードする遺伝子がホウ素過剰耐性に必須であることを明らかにしている。コンデンシンIIは細胞分裂時の染色体凝集を担うほか、ヒト、ショウジョウバエや酵母などでは転写調節やDNA損傷修復などの役割を担うことが知られている。動物などで知られているDNA損傷復機能に着目して、ホウ素毒性のプロセスとして、DNA損傷の可能性について検討し、シロイヌナズナ野生株においてDNA損傷修復遺伝子の発現を調べ、ホウ素過剰によりそれらの遺伝子発現が誘導されることを明らかにした。また、コメットアッセイによるDNA損傷の定量的解析を行い、ホウ素過剰によりDNA損傷が蓄積することを示した。これらの結果は、ホウ素過剰がDNA損傷を引き起こすこと意味している。くわえて、コンデンシンIIの変異株がDNA損傷を起こす試薬に対して感受性を示し、さらにDNA損傷修復遺伝子の発現量・DNA損傷の程度ともに通常の条件でもホウ素過剰の条件でも野生型株よりも多いことを明らかにした。以上のことから、シロイヌナズナのコンデンシンIIがDNA損傷修復あるいはDNA損傷防御の機能を持っていること、さらにそのような機能を通じてコンデンシンIIはホウ素毒性を抑制していると考えられた。

第二章ではホウ素過剰耐性にプロテアソームが重要であることを示している。プロテアソームは主にポリユビキチン標識された異常タンパク質や不要なタンパク質の能動的分解を行うことで、細胞周期の調節、DNA損傷修復など様々な生命現象を司る巨大タンパク複合体であが、これらタンパク質複合体の変異株は、他の無機ストレスである塩、カドミウムやヒ素などには感受性を示さず、コンデンシンII及びプロテアソームの特定の変異がもたらす感受性はホウ素過剰特異的であることが示唆された。プロテアソームは真核および原核生物にも広く保存されているが、植物では特定のサブユニットに複数のパラログ分子が存在し、植物では機能に応じた様々な組み合わせのプロテアソームが形成されていると考えられている。プロテアソームを構成する19S Regulatory Particle (RP)のサブユニットうち少なくともRPN2a、RPN8、RPT2a及びRPT5aがホウ素過剰耐性に必須であることが示された。一方、これらのパラログ分子であるRPN2b、RPN8b、RPT2b、RPT5bはホウ素過剰に必須ではなかった。このことから、4つのサブユニットが共通に関わるプロテアソームの機能がホウ素過剰耐性に必須であること、一方でパラログ間ではホウ素過剰における機能が異なることが示された。さらに、変異株のポリユビキチン化タンパク質の蓄積量について調査を行い、その結果、rpt5aではホウ素過剰によってポリユビキチン化タンパク質がより蓄積しており、プロテアソーム活性が低下していることが示された。次に、野生型株及びrpt5aからポリユビキチン化タンパク質を精製し、ホウ素過剰によって量が増えるもの、rpt5aでのみ蓄積するあるいは量が増えるものがあるかどうかを、iTRAQ試薬を用いたLC/MS/MSによる解析を行い、22種のタンパク質についてはホウ素過剰によってポリユビキチン化が促進されること、かつrpt5aで蓄積量が多いことが示された。以上のことから、特定のサブユニットを持つプロテアソームが、ホウ素過剰によって誘導される特定のポリユビキチン化タンパク質の分解制御を行うことでホウ素過剰耐性に貢献していると考えられた。

第三章ではプロテアソーム変異株の地上部の成長が亜鉛欠乏に対して野生型株よりも感受性になることを示し、その栄養特異性について検討を行っている。

以上本研究は植物のホウ素過剰耐性機構について、4つの新規遺伝子を単離しその役割を明らかにしたものであり、学術上、応用上貢献するところが少なくない。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。