Boron (B) requirement in plants was first described more than 80 years ago, and B availability in soil is an important determinant of agricultural production. B availability in soil is limited in many regions in the world, including Japan. Understandings of the molecular basis of the requirement and transport are necessary for improvement of agricultural production.

Introduction

B is an essential element for plants. The only function of B in plants proven at the molecular level is borate crosslinking of rhamnogalacturonan-II in cell walls (O'Neill at al., 2001). This crosslinking was shown essential for normal expansion of leaves.

B in neutral solution is mostly present as boric acid (H3BO3). Being a noncharged molecule, boric acid has a very high permeability coefficient for lipid bilayer, and it has long been believed that B is transported passively. However, in 2002, BOR1 was identified as a B transporter capable of transporting B against B concentration gradient (Takano et al., 2002). Furthermore, NIP5;1, an aquaporin-like gene, which encodes a B channel was found and NIP5;1 facilitated B transport across the membrane. NIP5;1 was required for normal plant growth at B deficiency and was upregulated by transcriptional level. NIP5;1 mRNA accumulation increased by 15-fold in root under the low B condition (Takano et al., 2006).

The primary objective of the present study was to understand physiological function of NIP6;1, the most similar gene to NIP5;1 in Arabidopsis thaliana. The regulatory mechanisms of NIP5;1 in response to B, and differential expression and function of NIP5;1 and NIP6;1 were also studied.

Chapter 1 Arabidopsis thaliana NIP6;1 is a boric acid channel that functions in preferential transport of B to young leaves

In addition to being the most similar to NIP5;1, NIP6;1 has identical amino acids in the "selectivity filter". The selectivity filter is amino acid residues shown to be involved in selection of substrates based on the structure and function. To investigate physiological function of NIP6;1 in B transport, NIP6;1 transcript level, cell and tissue specificity of NIP6;1, and plant growth and B concentration of NIP6;1 T-DNA insertion plants were examined.

NIP6;1 transcript levels in rosette leaves were higher than those in root in contrast to the NIP5;1 whose transcript accumulates predominantly in roots. NIP6;1 mRNA accumulation in rosette leaves increased by 1.5-fold under B limitation, while the response was not evident in roots.

To investigate the subcellular localization of NIP6;1, The construct for expression of GFP-NIP6;1 under the control of a cauliflower mosaic virus 35S RNA promoter was introduced into A. thaliana plants (P35S-GFP-NIP6;1). NIP5;1 is already known to be localized at plasma membrane. P35S-GFP-NIP5;1 was also introduced into A. thaliana plants as a control for a plasma membrane localization. GFP fluorescence in NIP6;1-expressing transgenic plants was localized at the extreme cell periphery with a similar pattern to that in NIP5;1-expressing transgenic plants at the root elongation zone, indicating that NIP6;1 was also localized at the plasma membrane as was the case of NIP5;1.

To verify whether NIP6;1 has a similar B transport property to NIP5;1, NIP6;1 was expressed in Xenopus oocytes. These NIP6;1 and NIP5;1-expressing oocytes showed significant swelling upon exposure to boric acid. B concentration determined by using ICP-MS was higher in NIP6;1 and NIP5;1-expressing oocytes than in uninjected oocytes, suggesting that NIP6;1 also facilitates boric acid in X. oocytes

To analyze the cell-type specificity of expression of NIP6;1, NIP6;1 promoter fragment containing the 5' UTR fused to the GUS reporter gene was introduced into A. thaliana plants. Strong GUS staining was observed in vascular bundles of nodal regions and immature young leaves, but no staining was observed in the mature old leaves. These patterns were identical both under high and low B supply.

To characterize the function of the NIP6;1 gene in A. thaliana, two independent mutant alleles, nip6:1-1 and nip6:1-2, containing T-DNA insertions in the first exon and forth exon of NIP6;1 coding region, respectively were obtained. At a late vegetative stage, plants supplied with 0.1?M B showed small young leaves in both T-DNA insertion lines compared with that in wild-type (Col-0), whereas the mature leaves of both T-DNA insertion lines were apparently normal at 0.1?M B supply. Both insertion lines grew similar to the corresponding wild-type plants when B was supplied at 100 ?M.

B concentrations of young leaves of nip6;1-1 and nip6;1-2 plants grown at 0.1 ?M B were significantly reduced compared with that of the corresponding leaves in Col-0 plants, whereas concentration in the old leaves were not different between the wild-type and the insertion lines. At 100 ?M B supply, there was no significant difference among the lines for B concentrations in both young and old leaves. These data demonstrate that NIP6;1 is required for expansion of young rosette leaves under low B supply.

Taken together, I demonstrated that NIP6;1 was a functional B channel required for preferential B transport to young leaves under the low B condition

Chapter 2 Promoter analysis of NIP5;1, a boron deficiency inducible gene, in Arabidopsis thaliana

To clarify NIP5;1 expression mechanism in response to B, a series of 5' deletions of the upstream promoter region were generated and fused to the GUS reporter gene followed by introduction into A. thaliana plants.

The GUS expression analysis under normal and B deficiency condition revealed the following

1. Region between-580/-562 is required for expression in root tips under B deficiency.

2. Region between-448/-400 is required for expression in root elongation zone under B deficiency.

3. Region between-400/-300 is required for expression in bulk roots under B deficiency.

In summary, this analysis indicated that B deficiency induction of NIP5;1 promoter activity is governed at least 3 distinct cis-acting elements and each cis-acting element is required for upregulation of particular portions of roots. This is the first example of a gene whose nutrient induction is regulated by three distinct cis-acting elements in different cell types.

Chapter 3 Isolation of a NIP5;1-related boron limitation responseless mutant in Arabidopsis thaliana

To identify a key regulatory protein(s) controlling the upstream signaling cascades of NIP5;1 in response to B, A. thaliana mutants were screened.

Seeds of transgenic plants carrying promoter NIP5;1-GFP were mutagenized with ethyl methanesulfonate (EMS) and M2 seedlings were used for screening. Thirty thousand M2 seeds were screened for lines with reduced level of GFP fluorescence under the low B condition and eight lines were isolated. Genetic analysis suggests that the phenotype is caused by a single nuclear locus. In the two lines, genetic linkage was found in the downstream of chromosome 5. Fine mapping determined that these mutants were located in 50 kb between the mxk3 marker and mqn23 marker corresponding to the MXK3 and MQN23 clone. To my surprise, in this mutant, NIP5;1 expression was reduced in root tips but not in the bulk roots. It was concluded that regulation of gene expression in response to B concentration was governed by at least 2 distinct mechanisms, one operating specifically in root tips, the other in the bulk of roots.

Chapter 4 Growth improvement of nip5;1 by disruption of NIP6;1

Both NIP5;1 and NIP6;1 encode a boric acid channel, but, as I established above, their roles are different. To verify phenotypes of double T-DNA insertion lines of NIP5;1 and NIP6;1, physiological and molecular analyses were conducted.

Shoot growth in nip5;1-1 x nip6;1-1(referred to as WKO1) and nip5;1-1 x nip6;1-2 (referred to as WKO2) mutant plants was higher than that in nip5;1-1 single mutant plants, but lower than that in nip6;1-1 and nip6;1-2 single mutant plants under the low B condition. B concentration in shoots in WKOs mutant plants was higher than that in nip5;1-1 single mutant plants, but lower than that in nip6;1-1 and nip6;1-2 single mutant plants under the low B condition. The mRNA accumulation of NIP5;1 in nip6;1-1 and nip6;1-2 mutant plants was similar to that in wild type, and also the mRNA accumulation of NIP6;1 in nip5;1-1 mutant plants was not different from wild type.

Among the phenotypes, it is intriguing to point out that in my system, the WKO plants grow better than the nip5;1-1 single mutant plants. In other words, mutation in NIP6;1 rescued growth defect of the nip5;1-1 mutant plants. Considering that both NIP5;1 and NIP6;1 are boric acid channels with different roles in whole-plant B physiology, it is generally assumed that WKO mutants will show severer phenotype than the single mutants. I repeated these experiments 3 times and similar results were obtained in each case. It is not possible to demonstrate mechanisms underlying this phenomenon at this time, but this is the first example of the rescue of the phenotype by mutation of genes with similar function. The present finding opened a new field of combinatorial genetics and a novel strategy for crop improvement.

Chapter 5 NIP5;1 polar localization and regulatory mechanisms in response to B in Arabidopsis thaliana

The construct for expression of GFP-NIP5;1 under the control of the promoter NIP5;1 was introduced into A. thaliana plants (promoter NIP5;1-GFP-NIP5;1). GFP fluorescence in the transgenic plants was localized at the distal side of plasma membrane in root epidermis cells.

GFP-NIP5;1 is predominantly accumulate in roots, but not in shoots in P35S-GFP-NIP5;1 transgenic plants. On the other hand, GFP-NIP6;1 is accumulated both in roots and shoots in P35S-GFP-NIP6;1 transgenic plants. Accumulation of NIP5;1 mRNA was highly increased in roots, but not in shoots in P35S-GFP-NIP5;1 transgenic plants. In contrast, mRNA accumulation of NIP6;1 was highly increased both in roots and shoots in P35S-GFP-NIP6;1 transgenic plants. The promoters used to drive these genes are identical and it is not likely that the difference is due to the promoter activity. These data strongly suggest that accumulation of NIP5;1 transcript is regulated in among tissues through a post-transcriptional mechanism.

In this chapter I demonstrated two novel regulatory mechanisms for NIP5;1 accumulation. One is distal localization in root epidermis. Considering the physiological role of NIP5;1 as an boron influx channel, it is reasonable that NIP5;1 is distally localized. The second finding is the post-transcriptional regulation of NIP5;1 transcript accumulation. These finding revealed that NIP5;1 is regulated by not only induction level under B deficiency, but also by mRNA accumulation level and protein level.

Conclusion

Through the analysis of NIP6;1 and NIP5;1, I established novel aspects and mechanisms of B transport in plants. I believe the findings described in the present thesis will be one of the important foundation for future develpoment.

Tanaka, M., Fujiwara, T. (2007). Physiological roles and transport mechanisms of boron: perspectives from plants. Pflugers Arch. DOI: 10.1007/s00424-007-0370-8 (published on line)

ホウ素は植物の生育に必須な微量元素である。本論文は、シロイヌナズナにおける新たな輸送機構を明らかにすると共に、ホウ素栄養条件による制御機構についての解析を行ったものである。

本論文は序章と5つの章からなる。序章では、以下のことを中心にホウ素研究の現状を述べている。ホウ素は、細胞壁を構成するペクチン質多糖ラムノガラクツロナンIIとホウ酸アニオンがエステル結合を形成し細胞壁の構造維持に重要な役割を果たしている(0'Neill at al.,2001)。ホウ素は水溶液中(pH7.0前後)で主にホウ酸の形で存在する(H3BO3)。ホウ酸は無電化の低分子であるため膜透過性が高いことから、植物体内でのホウ酸輸送は主に受動拡散によるものだと考えられてきた。しかしながら、近年BOR1が同定された。BOR1はホウ素の濃度勾配に逆らってホウ素を輸送するホウ素排出型のトランスポーターである。(Takano et al.,2002)。さらに、NIP5;1がホウ素チャンネルであり、ホウ素欠乏下において根からのホウ素の吸収に必須であることが明らかとなった(Takano et al.,2006)。

第一章では、NIP5;1遺伝子に最も相同性の高い遺伝子、NIP6;1のホウ素輸送について研究を行っている。アフリカツメガエルの卵母細胞の発現系を用いてホウ酸の輸送能についての解析を行ったところ、NIP6;1にホウ酸輸送活性が認められた。NIP6;1はシロイヌナズナにおいて細胞膜に局在し、地上部のなかでもとくに節、葉脈に存在していた。根と堆上部でmRNAが検出されたが、地上部の方が発現が強かった。また、ホウ素栄養による発現誘導は地上部で認められた。複数の独立に得られたNIP6;1遺伝子破壊株をホウ素欠乏条件で栽培したところ、葉の一部の生育が異常になった。この異常は通常のホウ素条件で栽培した場合には見られなかった。これらのことから、NIP6;1はNIP5;1同様ホウ酸チャンネルであり、ホウ素欠乏条件での植物の、特に若い葉や花茎の正常な生育に必須であることを結論付けている。

第二章ではNIP5;1プロモーター内の低ホウ素応答領域(シス配列)の同定を目的とした研究を行っている。。本研究でプロモーターdeletion解析を行った結果、1.-580/-562領域がホウ素欠乏下における根端の発現に必須であった。2.-448/-400領域がホウ素欠乏下におけう根の伸長領域の発現に必須であった。3.-400/-30領域がホウ素欠乏下における根の成熟領域の発現に必須であった。つまり、根のある特定の部分でのホウ素に応答した発現に、NIP5;1プロモーター内に存在する3つの異なる領域それぞれが独立して関与していることを明らかにしている。

第三章ではホウ素に応答したNIP5;1の転写制御に関わる新たな遺伝子を同定するためにpromoter NIP5;1-GFP形質転換体植物を変異原処理し、スクリーニングを行った。得られた変異株は、低ホウ素条件において根端でのみGFP蛍光が抑制されていた。マッピングの結果、第5番染色体のAt5g65030.1とAt5g65140.1.の領域、約50kb上に位置することがわかった。このことは、NIP5;1のホウ素に応答した根における制御機構は少なくとも2つの異なるメカニズムが存在することを示唆するものである。

第四章では、NIP5;1、NIP6;1、2つの遺伝子を破壊した遺伝子破壊株を作製し、その成長を解析した。NIP5;1,NIP6;1遺伝子二重破壊株を低ホウ素条件で栽培したところ、NIP5;1遺伝子破壊株よりも生育がよく、NIP6;1遺伝子破壊株よりも生育が悪かった。葉のホウ素濃度も植物の生育と同じ傾向を示し、この二重破壊株を低ホウ素条件で栽培じたときの葉のホウ素濃度はNIP5;1遺伝子破壊株の葉の濃度よりも高く、NIP6;1遺伝子破壊株の葉の濃度よりも低かった。つまり、NIP5;1の遺伝子破壊株においては、NIP6;1が生育抑制効果を持っていることを示した。同じ機能を持つ2つの遺伝子の破壊においては、一重の遺伝子の破壊よりもさらに生育が悪くなるという例は多数報告されているが、良くなるという新しい例を発見している。

第五章ではpromoter NIP5;1-GFP-NIP5;1形質転換植物により、NIP5;1は遠心側に局在していることを明らかにした。また、P35S-GFP-NIP5;1とP35S-GFP-NIP5;1の形質転換植物のmRNAの蓄積の比較により、NIP5;1の地上部と根でのmRNAの蓄積のパターンが異なり、根でのみmRNAの蓄積が増加した。つまり根と葉でのNIP5;1の制御機構が異なっていることを示している。

以上、本論文は、複数の画期的な成果を含み、植物のホウ素輸送の分子機構の理解を深めるとともに、ホウ素栄養に応じた植物の反応機構につ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.