Conquering the cool temperature damages caused by frequent cool weather in summer season
has been a big challenge in rice cultivation in Northern Japan. In 1993,
Japan suffered from the historic cool weather for these 100 years.
It became the catastrophic decrease in rice yield especially on the Pacific Ocean side of northern Japan by spikelet sterility.
These damages were caused by continuous cool weather during the booting stage, the cool sensitive stage.
In 1993, it was also confirmed that the decrease in the yield caused by cool temperature was correlated with
the increase in nitrogen level.
During the booting stage, cool temperature at the young microspore stage is
the most harmful to rice reproduction by repressing the pollen development.
Therefore, in this study, the effects of high nitrogen (High-N) on the cool temperature damage were
analyzed focusing on the pollen development.
Chapter I Effects of high nitrogen supply and cooling at young microspore stage on factors related to sterility
Effects of High-N and cooling (High-N-cooling) at the young microspore stage on the factors involved
in the sterility such as the numbers of microspores, engorged pollen grains, pollen grains on the stigma,
and the pollen germination ratio were analyzed with Hayayuki, an early-maturing japonica rice variety
which has been used for researches on cool temperature damage.
1. The number of microspores
The number of microspores was decreased by High-N and shading, indicating that the maximum numbers of
engorged pollen grains were also lowered.
After cooling at the young microspore stage, the numbers of microspores were further decreased.
2. The number of engorged pollen grains
The number of engorged pollen grains was markedly decreased by cooling at the young microspore stage.
These decreases were enhanced by High-N and shading.
These decreases in the number of engorged pollen grains were more notable than those of microspores.
As the number of engorged pollen grains was decreased, the fertility reduced.
3. The number of pollen grains shed on the stigma and pollen germination
Cooling decreased both the numbers of engorged pollen grains shed on the stigma and germinated
pollen grains on the stigma and High-N enhanced this decreases.
The numbers of engorged pollen grains on one stigma was over 90 regardless of High-N,
shading and cooling treatments, and 90 pollen grains was thought to be enough for setting a seed.
Interestingly, even in the plants that have the similar number of pollen grains shed on the stigma,
the fertility in cooled plants was lower, and it was much lower under High-N and shading.
These results suggested that pollen grains in cooled plants, which seemed to be normal, might have lower activity.
From then on, I focused on pollen viability under High-N and High-N-cooling.
Pollen germination and pollen tube elongation were notably suppressed by High-N-cooling.
In order to exclude the effect of stigma and to quantify the pollen viability,
the pollen germination was tested on the agar plate,
and conspicuous suppression of pollen germination by High-N-cooling was observed.
From these results, it was suggested that the enhanced suppression on pollen development and
pollen viability may be involved in the intensified reduction in fertility under High-N-cooling.
The suppression on pollen germination by High-N-cooling was notable and it was thought to have a direct effect on sterility.
Chapter II Proteome analysis of rice mature anthers
To clarify the physiological aspects of the damages on pollen grains caused by High-N -cooling,
protein expression patterns in mature anthers were observed by the comparative proteome analysis.
In mature anthers, more than 1000 protein spots were detected by CBB staining on 2-DE gel images.
However, only 11 spots whose densities were changed by High-N and High-N-cooling treatments were
observed and 7 out of 11 protein spots were identified as rice proteins.
Three were expansins (EXPs) which is involved in cell elongation.
Alpha-EXP18 (EXPA18) and beta-EXP1 (EXPB1) were increased by High-N.
EXPA18 was decreased and EXPB13 was increased by High-N-cooling.
Next, 3 proteins involved in stress responses, calcium-dependent protein kinase 11 (CDPK11),
putative aldehyde dehydrogenase (ALDH) and heat shock protein 82 (HSP82),
were increased by High-N.
CDPK11 and HSP82 were decreased by High-N-cooling.
Fructokinase II (FKII), which is involved in sugar metabolism, was increased by High-N-cooling.
From these results, it was suggested that changes in cell elongation and sugar metabolism may involved in damages in pollen grain.
As 3 proteins which involved in stress responses were increased by High-N,
it was also suggested that High-N itself may be a kind of stress.
Chapter III Gene expression analysis in rice mature anthers
From proteome analysis of mature anthers, 7 proteins which were changed by High-N and High-N-cooling were identified.
Then changes in gene expression patterns in mature anthers were examined.
From the similarities between the patterns of gene expression and protein expression,
changes in EXPA18, EXPB1, putative ALDH and FKII proteins in the anthers were assumed to be regulated at the transcriptional level.
It was notable that 3 of 7 changed proteins were EXPs.
EXPs have cell wall loosening activity, and it was assumed that EXPs might be involved in pollen grains swelling,
germination and pollen tube elongation, because cell wall loosening is thought to be essential for these processes.
Therefore, expression patterns of members of EXP gene family in the anthers under High-N and High-N-cooling were analyzed.
All 26 EXPA s and 16 EXPB s examined were expressed in the anthers except EXPA12 .
As a result, 18 EXPA s and 6 EXPB s were repressed under High-N-cooling.
Among these downregulated EXP s, EXPA18 , EXPA19 and EXP20 had high similarities in the amino acid sequences,
suggesting that these three genes may constitute a distinct functional gene subfamily related to the decrease
in the pollen germination ability caused by High-N-cooling.
In this study, I examined the effects of High-N and High-N-cooling on increased sterility in rice plant.
High-N enhanced the suppressions on pollen germination caused by cooling.
Focusing on the pollen germination, physiological aspects of mature anther were examined.
From the results of comparative proteome analysis and gene expression analysis,
it was revealed that a number of EXP s were downregulated by High-N-cooling.
EXP s act on cell wall loosening. Zea m1, a maize pollen protein which consists of at least 4 EXPBs, looses the silk cell wall.
It was thought that EXPs in rice pollen also have similar function.
As numbers of EXP s were repressed under High-N-cooling,
it appears that these EXPs may be involved in the enhanced decreases in pollen germination ratio,
and consequently, cool temperature damage under High-N-cooling. It was thought that an important finding was
obtained from this study about the effects of High-N on cool temperature damages in rice plants.
（和訳）
北日本の稲作において冷害の克服は重要な課題である．
1993年，日本はこの100年間における最大の冷害に見舞われ，特に北日本の太平洋側では壊滅的な被害を受けた．
これは穂ばらみ期の冷温による障害型冷害であったとされている．
また，従来窒素施肥による冷害の拡大が知られており，このことは1993年冷害においても確認された．
イネは穂ばらみ期のなかでも小胞子初期に最も冷温感受性が高く，
冷温による花粉の発育障害が障害型冷害の最大の要因であることが明らかにされている．
本研究では，冷温によるイネの不稔を窒素施肥が助長する要因を明らかにする目的で，
冷温による不稔の主要因とされている花粉の発育障害に注目して研究を行った．
第1章 多窒素・冷温が不稔に関連する要素に及ぼす影響
多窒素および遮光によって小胞子数は減少した．
このことは常温においても多窒素や遮光によって最大充実花粉数が減少することを示している．
小胞子初期の冷温によって小胞子数はさらに減少した．
２． 充実花粉数
充実花粉数は小胞子初期の冷温により顕著に減少し，多窒素および遮光によってその減少は助長された．
小胞子数の減少に比べて充実花粉数の減少ははるかに顕著であり，充実花粉数の減少に伴って稔実歩合が低下した．
３．受粉数と花粉発芽
柱頭上の受粉数と発芽花粉数はともに冷温によって減少し，その傾向は多窒素によって助長された．
しかしながら受粉数は多窒素・遮光・冷温区においても90以上で，稔実に充分な数であった．
受粉数が同程度であっても稔実歩合は冷温処理した場合の方が低く，多窒素区および遮光区ではさらに稔実歩合が低かった．
これらの結果から正常に見える花粉であっても多窒素，冷温，遮光によって活性が低下することが示唆された．
そこで，花粉の活性に着目して多窒素・冷温の影響を検討した．
柱頭における花粉発芽と花粉管伸長は多窒素・冷温によって抑制された．
次に，柱頭の影響を除き，花粉の活性を数値化するために，寒天培地を用いて発芽試験を行ったところ，
多窒素・冷温によって花粉発芽は著しく抑制されることが明らかとなった．
以上のことから，多窒素栽培における障害型冷害の助長は，
冷温による花粉の発育阻害と花粉の活性低下が多窒素条件下では著しく助長されることが要因の一つであることが示唆された．
多窒素・冷温による花粉発芽率の低下は顕著であり，不稔に直接的に影響すると考えられたので，花粉の活性低下に着目することにした．
第２章 成熟葯のプロテオーム解析
多窒素・冷温による花粉の障害の生理的な側面を明らかにするため，
比較プロテオーム解析を行い成熟葯のタンパク質発現パターンを検討した．
成熟葯の二次元電気泳動像をCBB染色すると1000以上のタンパク質スポットが検出された．
しかし，11のタンパク質スポットのみが多窒素および多窒素・冷温によって変動し，7個がイネのタンパク質として同定された．
3個は細胞伸長に関与するエクスパンシン(EXP)であった．
alpha-EXP18 (EXPA18)とbeta-EXP1 (EXPB1)は多窒素によって増加した．
EXPA18は多窒素・冷温によって減少し， EXPB13は増加した．
次に，calcium-dependent protein kinase 11 (CDPK11)， putative aldehyde dehydrogenase (ALDH) ，
そしてheat shock protein 82 (HSP82) の3個がストレス反応に関与するものと分類され，
これらは全て多窒素によって増加し，CDPK11とHSP82は多窒素・冷温によって減少した．
糖代謝に関与するfructokinase II (FKII)は，多窒素・冷温によって増加した．
これらの結果から細胞伸長や糖代謝における変動が花粉の障害に関与することが予想された．
またストレス反応に関与するタンパク質の増加から，多窒素そのものがストレス要因かもしれないと考えられた．
第３章 成熟葯の遺伝子発現解析
成熟葯のプロテオーム解析から多窒素・冷温によって変動する7個のタンパク質を見出したので，
これらのタンパク質の遺伝子発現を検討した．
遺伝子発現とタンパク質発現パターンの類似性から，成熟葯におけるEXPA18，EXPB1，putative ALDH，FKII タンパク質の
発現は転写レベルで制御されていると考えられた．
同定された7個のタンパク質のうち，3個がEXPであったことは注目に値すると考えた．
EXPは細胞壁をゆるめる働きをもつので，花粉の肥大，発芽，花粉管伸長など，
細胞壁を緩めることが必須と考えられる過程に関与すると推測される．
そこで，成熟葯における EXP 遺伝子ファミリーの発現パターンを検討した．
試験に用いた26個の EXPA と16個の EXPB のうち， EXPA12 を除くすべての EXP の発現を認めた．
多窒素・冷温によって18個の EXPA と6個の EXPB の発現の低下を認めた．
発現が低下した EXP の中でも， EXPA18 ，EXPA19 ，EXP20 はアミノ酸配列において高い類似性を示したので，
これらは，多窒素・冷温による花粉発芽活性の低下に関係した機能を持つ，
特徴的なサブファミリーを形成しているのかもしれないと考えた．
本研究では多窒素施肥が冷温障害を助長する要因について検討した．
冷温による花粉発芽の抑制が多窒素によって著しく助長されたことから，
花粉発芽に注目して成熟葯について生理的側面を検討した．
比較プロテオーム解析および遺伝子発現解析から，
多くの EXP 遺伝子の発現が多窒素・冷温によって抑制されることが示された．
EXPは細胞壁を緩める働きを持つ．トウモロコシ花粉に存在するZea m1は少なくとも４つのEXPBから成り，
柱頭細胞を緩めることがわかっている．
イネ花粉の EXP も類似の働きを持つと推測される．
多窒素・冷温によって多くの EXP 遺伝子の発現が抑制されたことから，これらの EXP は花粉発芽率の低下に関与し，
結果として多窒素栽培における冷温障害の助長に関係することが示唆された．
以上のように，本研究では多窒素による冷温障害の助長について重要な知見を得られたものと考えられる．
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