Increased salinization of arable land is greatly reducing yield much below the genetic potential of the cultivated plants. Consequently, new strategies including both genetic manipulation and traditional breeding approaches represent a major research priority to enhance crop yield stability on saline soils. Therefore, an understanding of physiological and genetic mechanisms how plants tolerate and acclimate to saline environments is of great importance for genetic modification and plant breeding. Since the capacity of plants to maintain a high cytosolic K+/Na+ ratio is one of the key determinants of plant salt tolerance, identification of K+ and Na+ transport systems would lead to a better understanding about salinity tolerance mechanisms. Plants differ greatly in their tolerance of salinity, as reflected in their different growth responses. Most crops are glycophytes, thus are not capable of growing' under high saline conditions. Halophytes, plants adapted to saline habitats, have evolved various mechanisms to overcome salt stress in the long-term natural selection. Therefore, understanding the salt-tolerance mechanism(s) of monocotyledonous halophytes will aid in improving the salt tolerance of cereals. This study focused on the isolation and characterization of K+ and Na+ transport genes from a halophytic plant, Puccinellia tenuiflora (P. tenuiflora).

(1) Cloning of a high-affinity K+ transporter gene PutHKT2;1 from P. tenuiflora and its functional comparison with OsHKT2;1 from rice in yeast and Arabidopsis

HKT-type transporters have been characterized in several plant species and the known plant HKTs have been shown to perform diverse functions. TaHKT2;1 functions as a Na+-uptake pathway in wheat roots, while rice OsHKT2;1 mediates Na+-uptake into K+-starved roots. In Arabidopsis, AtHKT1;1 functions in retrieval of Na+ from the transpiration stream. These previous studies showed the important role of HKT transporters in plant salt tolerance.

In this study, a high-affinity K+ transporter PutHKT2;1 cDNA from the salt-tolerant plant P. tenuiflora was isolated. PutHKT2;1 belongs to subfamily 2 in HKT-family by a phylogenetic analysis. By using the green fluorescent protein (GFP), the PutHKT2;1 protein was shown to localized in the plasma membrane. Expression of PutHKT2;1 was induced by both 300 mM NaCI and K+-starvation stress in roots, but only slightly regulated by those stresses in shoots. PutHKT2;1 transcript levels in 300 mM NaCI were doubled by depletion of potassium. Yeast transformed with PutHKT2;1, like those transformed with PhaHKT2;1 from salt-tolerant reed plants (Phragmites australis), (i) were able to take up K+ in low K+ concentration medium or in the presence of NaCI, and (ii) were permeable to Na+. This suggests that PutHKT2;1 has a high affinity K+-Na+ sym port function in yeast. Arabidopsis over-expressing PutHKT2;1 showed increased sensitivities to Na+, K+, and Li+, while Arabidopsis over-expressing OsHKT2;1 from rice showed increased sensitivity only to Na+. In contrast to OsHKT2;1, which functions in Na+-uptake at low external K+ concentrations, PutHKT2;1 functions in Nat-uptake at higher external K+ concentrations. These results show that the modes of action of PutHKT2;1 in transgenic yeast and Arabidopsis differs from the mode of action of the closely related OsHKT2;1 transporter.

(2) Cloning of an AKT1-type K+-channel a subunit gene PutAKT1 from P. tenuiflora and its functional analysis in Arabidopsis

Plant voltage-dependent K+ channels have been found to play an important role in K+ homeostasis in higher plants. Channel-mediated K+ uptake at the soil-root interface in all plants studied to date has been associated with homologues of the Arabidopsis thaliana AKT1 (Arabidopsis K+ transporter 1) channel. Previous studies demonstrated the adverse effect of Na+ on the K+ uptake through AKT1-type channel, indicating that the regulation of AKT1-type channel is one important determinant to salt stress tolerance.

In this study, cDNA for a subunit of an inward-rectifying K+ channel was isolated from the salt tolerant P. tenuiflora and designated as PutAKT1. The phylogenetic analysis showed that PutAKT1 belongs to AKT1-subfamily in Shaker K+ channel family. PutAKT1 was localized in the plasma membrane and it was preferentially expressed in the roots. The expression of PutAKT1 was induced by K+-starvation stress in roots and was not down-regulated by the presence of excess Na+. Arabidopsis plants over-expressing PutAKT1 showed enhanced salt tolerance compared to wild-type (WT) plants. Under 75 mM NaCI stress, the PutAKT1-expressing plants showed better shoot phenotype and higher fresh- and dry-weight than that of the WT. Furthermore, by the expression of PutAKT1, the K+ content of Arabidopsis increased under normal, K+-starvation, and NaCI-stress condition. Arabidopsis expressing PutAKTI also showed a decrease in Na+ accumulation both in shoot and root. These results suggest that (i) PutAKT1 is involved in mediating K+ uptake in both low- and high-affinity uptake range, and (ii) unlike its homologues in rice, it seems to mediate K+ uptake even under salt stress condition.

(3) Functional comparison of K+ channel a and β subunit of rice and P. tenuiflora: The role of K+ channel a and β subunit interaction in K+-nutrition

Plant voltage-dependent K+ channels are multimeric proteins which are composed of homotetrameric a subunit and an auxiliary β subunit. The (3-subunits are not required for the K+ channel to be active. However, they can confer different properties to the channel. Some β-subunits have been shown to change the inactivation rate of their target channels whereas other behave as chaperones promoting channel maturation and increased the stability of K+ channel protein on the plasma membrane.

We cloned a cDNA for K+ channel 13 subunits from the P. tenuiflora and named it KPutB1. KPutB1 was preferentially expressed in the roots of P. tenuiflora. Potassium starvation, 300 mM NaCI stress, or the combination of both stresses lead to a transient induction of KPutB1 transcript levels in both roots and shoots. For further analysis, the K+ channel a and β subunit homologues from rice (OsAKT1 and KOB1, respectively) were also isolated. By yeast two-hybrid assay we demonstrated that KPutB1 interacts with PutAKT1. We also found that the interaction between K+ channel a- and 13-subunits could occur across plant species, as KPutB1 could also interact with rice OsAKT1 and Arabidopsis AKT1. In order to understand the functional relevancies of this interaction on K+-nutrition, co-expression experiments in yeast were conducted under various ionic conditions. Our result showed that yeast co-expressing PutAKT1 and the β subunits (KPutB1 and KOB1) had a better growth and higher K+-uptake ability than yeast expressing PutAKT1 alone. In contrast, co-expressing the β subunits (KPutB1 and KOB1) with OsAKT1 leads to a reduction in the yeast growth and K+ uptake rate in comparison to that of yeast expressing OsAKT1 alone. These results suggest that (i) co-expressing K-channel a and R subunit in yeast would lead to a different growth and different K+-uptake ability than yeast expressing K-channel a subunit alone, and that (ii) the growth phenotype of the co-expressing yeasts was dependent on the a subunit component. Arabidopsis plants over-expressing K+-channel R subunit of P. tenuiflora or rice showed increased shoots K+ content and decreased roots Na+ content under control, 75 mM NaCI, and K+ starvation stress conditions. However, the different ion accumulation between WT and K+-channel subunit-expressing plants does not lead to any phenotypic difference with the WT under salt stress and K+ starvation conditions.

It was already shown that the salinity tolerance of P. tenuiflora depends on its ability in maintaining K+ uptake and limiting the unidirectional Na+ influx under salt stress. A low-affinity K+ uptake system that selectively transports K+ in P. tenuiflora roots have been reported to play important role in the salinity tolerance of this plant, and PutAKT9 is the candidate of this low-affinity K+ uptake system. KPutB1 may increase the activity of PutAKT1 and help the K+ channel to function properly even under unfavorable condition such as salinity. The shoot Na+ content of P. tenuiflora was reported to remain relatively unchanged under salt stress, and our results suggest that PutHKT2;1 may involved in maintaining Na+ homeostasis of this plants (e.g. by retrieving Na+ from the transpiration stream). The mechanism of salinity tolerance is a very complex phenomenon and it involves multiple stress responsive genes. Detailed analysis of each gene and also the cross talk within components of stress signal transduction pathway are of great importance. Careful utilization of specific genes, including targeting to specific cell types or tissues, should help in developing salt tolerant cultivar.

世界各地で拡大している塩類集積土壌により耕地面積が減少している。耕地面積の減少を食い止めるためには耐塩性作物を育種することが必要であるが、そのためには植物の耐塩性機構を明らかにする事が重要である。植物の耐塩性の指標として、高濃度の塩存在化でK+/Na+値を高く保つ能力がよく用いられる。したがって植物におけるK+およびNa+の輸送機構を明らかにする事は、耐塩性機構を理解する上で非常に重要な課題である。

一般的な作物は中性植物であり、高濃度の塩存在化では著しい生育阻害を受ける。それに対して塩類集積土壌に生育できる塩性植物が多数存在し、そのような植物の耐塩性機構を明らかにする事により、作物の耐塩性を改良する上で有用な知見が得られる事が期待できる。本研究では、高濃度塩存在化でK+/Na+値を高く維持する能力に由来する極強の耐塩性を有する野生植物Puccinellia tenuiflora (P. tenuiflora)を用いて、K+およびNa+の輸送に関与するタンパク質の遺伝子に関する詳細な解析を行った。

1章の緒論では、研究の背景、意義と目的について述べている。

2章では、P. tenuifloraとイネからHKT(a high-affinity K+ transporter)タイプのカリウムトランスポーター遺伝子(PutHKT2;1およびOsHKT2;1)を単離し、酵母と形質転換シロイヌナズナを利用した比較機能解析を行った。GFPを用いた解析からPutHKT2;1は細胞膜に局在することがわかった。遺伝子発現は根で特に強く、しかも高濃度の塩ストレス(300 mM NaCl)およびK+飢餓により発現が誘導された。PutHKT2;1を導入した酵母を用いた実験によりPutHKT2;1はOsHKT2;1とは異なり高アフィニティー型のNa+-K+供輸送体としての機能を持つ事が明らかになった。PutHKT2;1を高発現するシロイヌナズナは、Na+, K+, and Li+に対して感受性をもち、OsHKT2;1を高発現するシロイヌナズナがNa+だけに感受性になるのとは異なっていた。また、PutHKT2;1導入シロイヌナズナとOsHKT2;1導入シロイヌナズナとは、外部K+濃度に応じたNa+吸収能力の点で大きな違いがある事がわかった。

3章では、電位非依存型のK+チャンネルをコードする遺伝子(PutAKT1)をP. tenuifloraから単離し、解析した。PutAKT1はShaker K+チャンネルファミリーのAKT-1サブファミリーに属すことがわかった。またPutAKT1は細胞膜に局在し、遺伝子発現は根で強く観察された。その遺伝子発現はK+飢餓で誘導されるとともに、他の植物で見られるような高濃度Na+による発現抑制を受けなかった。PutAKT1を高い発現する形質転換シロイヌナズナは、野生型と比較して塩ストレス条件下(75 mM NaCl)で植物体の重量が大きい事から、より強い耐塩性を持つ事がわかった。また形質転換体では、通常条件、K+飢餓条件および塩ストレス条件のいずれにおいても、植物体内K+含有量が野生型よりも高くなり、逆にNa+含有量は低下していた。これらの結果からPutAKT1は外部K+濃度に関わらずK+の輸送に関与し、その機能は外部Na+濃度に影響を受けない事が明らかになった。

4章では、AKT1タンパク質(K+チャンネルのα-サブユニット)と相互作用し、K+チャンネルの安定性等に関与する事が知られているβ-サブユニットをコードする遺伝子をP. tenuifloraおよびイネから単離し(KPutB1およびKOB1)、比較機能解析を行った。KPutB1の遺伝子発現は、K+飢餓および高濃度Na+存在下で上昇した。KPutB1が PutAKT1と相互作用する事は酵母two-hybrid法により確かめられた。またKPutB1はイネのOsAKT1やシロイヌナズナのAKT1とも相互作用する事がわかった。これらの相互作用の意義について明らかにするために酵母にα-およびβ-サブユニットを同時に導入する実験を行った。その結果、PutAKT1をβ-サブユニット(KPutB1 またはKOB1)と同時に酵母に導入すると、酵母の生育とK+吸収能力がPutAKT1だけを導入した場合より改良されるのに対し、OsAKT1をβ-サブユニットと同時に酵母に導入するとそれらは低下した。また、KPutB1またはKOB1を高発現する形質転換シロイヌナズナでは、塩ストレス(75 mM NaCl)およびK+飢餓条件下で野生型と比較して植物体地上部のK+含有量が上昇し、根のNa+含有量は低下した。しかし形質転換体の耐塩性等には野生型との差は観察されなかった。

以上のように、本研究では耐塩性極強野生植物P. tenuifloraを用いて、K+およびNa+の輸送に関与するタンパク質の遺伝子をクローニングし、イネ等の相同遺伝子との比較から、それらの遺伝子がP. tenuifloraの有する、「高濃度塩存在化でK+/Na+値を高く維持する能力」に関与する事を明らかにした。このような知見は耐塩性に関する分子育種を行う上で非常に重要であり、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。