Tomato(Lycopersicon esculentum Mill), the second largest producing vegetable crop in the world, is known as a chilling-sensitive plant. To understand the physiological and molecular bases in chilling tolerance in tomato, agronomical characteristics under field conditions and their chilling responses at various growth stages F₁ hybrid(Nitaki-Koma). In some cases, a wild tomato accession (Lycopersicon hirsutum LA 1777), which shows greater tolerance to chilling temperatures, was employed to further understand the mechanism of chilling tolerance. Specifically, in vitro seed germination, seedling growth, accumulation of sugars and proline, expression of a low-temperature-induced transcription factor (LeCBFs) and some cold-responsive genes were characterized. Furthermore, a novel CBF like gene (LhCBF1) of the wild tomato accession was cloned and sequenced, and its expression pattern in leaves during chilling was studied. With these, the molecular basis of the differences in chilling tolerance in the tomato cultivars was discussed to use the tomato germplasm to produce chilling tolerant cultivars.

　Agronomical characteristics of the tomato cultivars: The maternal parent "P1" was superior to the paternal parent "P2" in the agronomical characteristics. The F1 hybrid showed the hybrid vigor and high-parent or mid-parent heterosis in all of the agronomical characteristics especially in the total number of fruits/plant and the total plant fruit production. Determination of the percentage of dry weight in the 12-week-old tomato plants showed that all of the cultivars had similar percentages of dry weight.

　Effect of chilling on tomato seed germination and seedling growth: Seeds of the three cultivars along with the wild tomato accession germinated with no apparent disturbance under warm temperatures. However, under chilling stress conditions (23℃ after 2℃ pre-treatment or 10℃), seeds of P1, F₁ and the wild tomato germinated faster than those of P2. In addition, seedlings of those genotypes grew much better under chilling stress than those of P2. The P1, F₁ and the wild tomato seedlings showed high abilities of recovery and survival after chilling and rewarming.

　Chilling tolerance of tomato plants: When whole plants were chilled at different temperatures, P1 and F₁ cultivars were more chilling tolerant than P2 plants. On the other hand, the wild tomato accession showed the highest chilling tolerance comparing with the cultivated tomatoes. When both the cultivated and wild tomatoes were severely chilled at 0℃ for 2 weeks, the wild tomato plants could survive but all of the cultivated tomato cultivars died.

　Effect of chilling on electrolyte leakage from tomato leaves: Electrolyte leakage from leaves was determined to evaluate the chilling tolerance of the leaves during incubation at 2℃ for 11days or at 5℃ for 21days, P1 plants chilled at 2℃ and rewarmed had lower extent of electrolyte leakage at day 1 and 3 than that of P2 and F₁ plants. In all plants,prolonged chilling treatments resulted in very high percentages of electrolyte leakage(88-100%) both after chilling and rewarming treatments.

　When chilling temperature was raised to 5℃, the extent of electrolyte leakage became much less than that of leaves chilled at 2℃. Again, P1 plants had less leakage than P2 or F₁ plants when chilled for a longer period(e.g., 21days). Thus, these results collectively indicate that P1 leaves seem to be much stronger against chilling temperatures than P2 or F₁ leaves, which is consistent with the results of chilling tests with whole plant systems. Interestingly, electrolyte leakage was almost similar between the wild tomato accession (chilled at 5℃) and the cultivated tomatoes although there is a considerable difference in chilling tolerance between them.

　Effect of chilling on sugar content: Effect of chilling treatment on sugar(sucrose, glucose, and fructose) contents were determined using 6- and 8-week-old tomato plants of P1,P2, and F₁ cultivars chilled at 5℃ for 21days. All of the three cultivars contained similar amounts of sugars before chilling. Upon chilling, the content of the three sugars increased gradually in the three cultivars. P1 and F₁ (the chilling tolerant cultivars) accumulated sugar in their leaves much more than that of P2(the chilling sensitive cultivar). On the other hand, sugar contents in the leaves of non-chilled wild tomato accession plants was greater than that of the cultivated tomato cultivars; about four times in sucrose and about two times in glucose and fructose. However, upon chilling the sugar content did not increase much in leaves of the wild tomato accession.

　Effect of chilling on proline content: Proline content in leaves of 8-week-old plants was statistically greater (P＜0.05) in P1 than in P2 or F₁ before chilling treatment. A significant increase in proline amount in leaves occurred after 2 weeks of chilling at 5℃. There was only a small difference in the amount among the three cultivars. With the wild tomato accession, proline content in leaves before chilling was less than that of the cultivated tomatoes. Proline content then increased up to 2 folds after 1 day of chilling and remained stable until 7 days. However, proline content increased again to reach about 6.4-and 11.2-fold than that of the non-chilled plants after 14 and 21 days of chilling, respectively.

　Induction of the CBF and the CBF-related cold-responsive genes by chilling: The Northern blot analyses revealed that exposure of plants to a low temperature at 5℃ resulted in an induction of only LeCBF1 but not LeCBF2 or LeCBF3 in all of the three tomato cultivars. Upon chilling at 5℃,LeCBF1 transcript in P1 leaves was detected at 15min and accumulated gradually up to 2hours, but decreased thereafter. In P2,LeCBF1 transcript was barely detectable before chilling but increased gradually up to 1 hour and then decreased thereafter. In F₁, the accumulation of LeCBF1 transcript was noticeable after 1 hour of chilling. Furthermore, chilling-induced expression of LeCBF1 was greater in P1 than in P2 or F₁. When chilling temperatures were raised to 10 or 15℃, the expression of LeCBF1 decreased in general in all tomato cultivars. At 15℃, there was little or no induction in LeCBF1 in all three cultivars during the treatment. This shows a reverse correlation between low temperature and expression of LeCBF1 gene.

　The expression of some cold-responsive genes encoding a putative proteinase inhibitor(TC115995), a glycine-rich protein(TC117384), a dehydrin-like protein(TC116174) and a homolog of a potato dehydrin Ci7(TC116013) before and after chilling at 5℃ was greater in the chilling tolerant tomato cultivars(P1 and F₁) than the chilling sensitive one(P2). The dehydrin Ci7 gene homolog was expressed only in the leaves of P1 and F₁ chilled plants. However, the role of these cold-responsive genes in acquiring the chilling tolerance of the tomato cultivars is still to be determined.

　Isolation and sequencing of a nevel CBF like gene(LhCBF1) in leaves of the wild tomato accession: A cDNA was constructed from leaves of the wild tomato plants chilled at 5℃ for 1 hour. Sequence of one clone for LhCBF1 showed a nucleotide sequence of 932bp(accession number: AB231689). The amino acid sequence deduced from LhCBF1 cDNA contained 222 amino acids. Furthermore, there was a high degree of homology(90.99%identity) between LhCBF1 and the cultivated tomato LeCBF1. Different degrees of homology were observed with LeCBF2 and LeCBF3 from tomato, CaCBF1b from pepper, BnCBF1 from canola and AtCBF1 and AtCBF3 from Arabidopsis. The Southern blot analysis showed multiple fragments hybridized with the LhCBF1 probe, suggesting the presence of the LhCBF1 gene family. The RT-PCR analysis showed that the LhCBF1-like gene was induced in the wild tomato plants in response to low temperature at 5℃. Although it was expressed before chilling, the expression increased after 30 minutes of chilling, reached gradually to the maximum after 4 hours, and then began to decrease thereafter. After 24 hours, it was completely desppeared. This was the first record of the CRT/DRE binding factor in the chilling tolerant wild tomato L.hirsutum LA1777. The LhCBF1 will provide an efficient tool to increase chilling tolerance of tomato plants.

（和訳）

　トマト(Lycopersicon esculentum Mill) は、世界第２位の生産量を持つ野菜で、冷温感受性植物である。 トマトの冷温耐性に関わる生理学的、分子生物学的基盤を理解することを目的に、圃場における園芸学的特徴と 実験室内における様々な成長ステージでの冷温耐性を２品種(Coudoulete "P1" and Piline "P2")と その交配雑種(Nitaki-Koma"F₁")を用いて調べた。 さらに、冷温耐性が高い野生型トマト(Lycopersicon hirsutum LA1777)を用いて比較を行った。 具体的に以下に述べる８項目の研究を通じて、トマトにおける冷温耐性の違いにおける詳細な解析を行い、 より冷温耐性を持つ品種を育成する基盤を構築した。

　栽培トマト品種の農業的特徴： 母親であるP1は父親であるP2に比べ、全ての農業形質において優れていた。 交配雑種F₁は、全ての調べられた形質において、雑種強勢や両親の平均値よりは高い値を示した。 特に、植物個体あたりの果実数や果実収量で顕著に見られた。 また、速物個体の乾重量は、３品種で違いは見られなかった。

　種子発芽と実生の生育に対する冷温の効果： 常温における発芽や実生生育に関しては、供試した３栽培品種と野生型との間に違いは見られなかった。 しかし、冷温条件下（２℃処理後に２３℃で測定、あるいは、１０℃で測定）では、P1とF₁、および、 野生型トマトがP2に比べて優れた発芽と生長を示した。 また、一旦冷却して常温に戻した場合の実生成長も、これらの差異が明確に認められた。

　植物体レベルにおける冷温耐性： 種子および実生の冷温耐性と同様に、P1とF₁植物体はP2植物体に比べ、高い冷温耐性を示した。 野生型トマトは、これらの冷温に比較的強い栽培型トマトに比べさらに耐性があり、０℃で ２週間処理するという非常に厳しい条件に曝した場合でも生き残り、常温に戻すと成長を再開した。

　単離葉レベルにおける冷温耐性（電解質漏失法）： ２℃という厳しい条件で冷温処理を行うと、短い処理期間（１～３日）では、P1植物がP2やF₁に比べ、 傷害度が小さかった。 しかし、それ以上冷温処理を継続すると、全ての材料が傷害を受けた。 処理温度を５℃に上げると、傷害を受ける度合いは小さくなったが、やはりP1がP2やF₁に比べ耐性があった。 ただし、これは、長期間に渡って冷温処理を行った場合である（２１日間）。 この条件下で、高耐性を持つ野生型と栽培型の電解質漏失度は差がなかった。

　糖含量における低温の効果： ３栽培品種間で冷温処理前には糖含量に差は見られなかったが、冷温処理（５℃）とともに糖含量が大きく増大した。 冷温耐性が高いP1とF₁では、耐性の低いP2に比べ糖の増加度が大きかった。 一方、野生型トマトは、冷温処理前から多量の糖を含んでおり、冷温に応答した糖含量の増加はそれほど顕著ではなかった。

　プロリン含量における低温の効果： 冷温処理前にも、P1のプロリン含量はP2やF₁に比べ大きかった。 ５℃の冷温処理２週間では、プロリン含量は増加したが３品種間での違いは見られなかった。 野生型トマトにおいては、冷温処理前の含量は栽培型より少なく、冷温処理中にもその増加速度は遅かった。 しかし、２，３週間処理を続けるとプロリン含量は増加することから、栽培型トマトとは異なった応答経路が 存在すると考えられる。

　冷温処理による低温誘導性転写因子(CBF)とその標的遺伝子の誘導： ５℃の冷温処理は、低温誘導性転写因子のうちLeCBF1の誘導を引き起こすが、 LeCBF2やLeCBF3は誘導されない。 LeCBF1の低温誘導性発現はP1で最も顕著で、その程度も大きかった。 しかも、発現誘導が長く継続した。 P2では発現誘導は起こるが、その期間は短く、冷温処理を１時間以上続けると発現が急速に減少した。 F₁では短期間の誘導が見られた。 処理温度を１０℃あるいは１５℃で行うと、LeCBF1の誘導の程度は急速に減少した。 LeCBF1の標的遺伝子と報告されている４遺伝子の冷温処理期間中の発現を調べたところ、 いずれも、耐性の高い品種（P1およびF₁)で高く見られ、特に、 dehydrin Ci7 homolog遺伝子はP2での発現が確認できなかった。 今後、これらの低温誘導性遺伝子発現と冷温耐性の関係を詳細に調べる必要がある。

　野生型トマトからの新規CBF様遺伝子(LhCBF1)の単離とその発現解析： 冷温処理を行った野生型トマトの葉からcDNAライブラリーを作成し、各種の植物で報告されている CBF遺伝子の保存領域配列を用いて、LhCBF1を単離した。 LhCBF1cDNA(accession number:AB231689)は開始コドンを含む９３２塩基対からなり、 ２２２アミノ酸からなるタンパク質をコードしていた。 いくつかの保存領域を確かに含んでおり、トマトLeCBF1とは高い相同性を有していた。 他の植物（ピーマン、カノーラ、シロイヌナズナ）で報告されているCBF遺伝子とも高い相同性があった。 サザン解析はLhCBF1が遺伝子ファミリーを形成していることを示唆した。 一方、ノーザン解析はLhCBF1が冷温処理なくても発現していることや、冷温処理によって さらに発現が誘導され、４時間後に最大になることを示した。 しかし、２４時間後には発現がなくなった。 野生型トマトからのCBF様遺伝子の報告はこれが初めてであり、今後、トマトの冷温耐性増大に 有効に使用できると考えられる。