IbTLD modulated reactive oxygen species scavenging and DNA protection to mediate salinity stress tolerance

TLDc是在2002年被發現含有Tre2/Bub2/Cdc16 (TBC) 與lysin motif (LysM) 的蛋白質功能區塊，TLDc相關的蛋白質可能與由過量活性氧化物 (ROS) 所引起之氧化逆境的防禦有關，而植物中ROS (包含H2O2與O2-) 的含量則會受到鹽逆境或是其他許多環境的改變而增加，但是，目前對於TLDc相關蛋白的調控機制尚不清楚。在本研究中，利用會大量表現甘藷 (Ipomoea batatas) TLDc基因 (IbTLD) 的轉基因菸草來觀察IbTLD在鹽逆境與耐受力所扮演的角色。在鹽逆境下，TLD轉殖株 (TLD-1與TLD-2) 具有較野生型 (W38) 更好的發芽率、葉綠素含量與根長表現。除此之外，利用Gene Ontology (GO) term分析RNA sequencing的結果，進而了解TLDc相關的下游基因調控網路，結果顯示在鹽逆境下轉殖株有許多與逆境反應、DNA保護和修復的基因會受到逆境所誘導。在RT-qPCR也顯示轉殖株中的NtFeSOD3.2、NtAPX3、NtNUDT10L與NtNUDT9L的表現量都比W38還要高。在一般情況與鹽逆境下，轉殖株的抗氧化酵素抗壞血酸過氧化酶 (ascorbate peroxidase, APX) 活性較W38的還要高，且在鹽逆境下，轉殖株的過氧化氫 (H2O2) 與丙二醛 (malondialdehyde，MDA) 含量都較W38的還要低。在轉殖株中，鹽逆境所導致的DNA ladder也有受到降低。因此推測，IbTLD可能會調控許多逆境相關基因的表現來清除ROS以及保護DNA，進而提高植物對於鹽逆境的適應力。TLDc domain, a novel protein functional domain containing Tre2/Bub2/Cdc16 (TBC) and lysin motif (LysM) domain, was identified in 2002. TLDc-related protein might be involved in the defense of oxidative stress caused by excessive reactive oxygen species. The amount of ROS, including H2O2 and O2−, in plants can be changed upon the different external environments including salt stress. However, the regulatory mechanism of TLDc-related protein is still unknown. In this study, transgenic tobaccos overexpressing Ipomoea batatas TLDc gene (IbTLD) were created to characterized the roles of the IbTLD in tolerance of salt stress. Under salt stress, transgenic lines showed better germination rates, chlorophyll contents and root lengths than wild type (W38) . In addition, RNA sequencing (whole transcriptome shotgun sequencing) incorporating with Gene Ontology (GO) term analysis was used to investigate TLDc-related downstream genes network. Results revealed several genes involved in responses of stress and protection and repair of DNA were influenced. Under normal and salt stress conditions, quantitative reverse transcription PCR also presented expression levels of NtFeSOD3.2, NtAPX3, NtNUDT10L and NtNUDT9L in transgenic lines were elevated compared to those in W38. The transgenic lines showed higher ascorbate peroxidase (APX) activity than W38 under normal and salt stress conditions compared to W38. Besides, transgenic lines also showed lower hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents under salt stress conditions. The salt-induced DNA ladder in transgenic lines was also deceased. Therefore, IbTLD might regulate the expression of stress related genes to mediate scavenging of ROS and protecting of DNA. Further, the abilities of adaptation to salt stress were enhanced.致謝 i 摘要 ii Abstract iii 目次 iv 表目次 vi 圖目次 vii 前言 1 1. 植物與非生物逆境以及氧化逆境 1 2. Reactive oxygen species (ROS) 活性氧化物 3 3. TLDc功能區塊 (TLDc domain) 5 4. 次世代定序技術中RNA sequencing的應用 5 5. 研究目的與方向 6 材料與方法 7 1. 菸草 7 2. H2O2處理 7 3. 鹽處理 7 4. 次世代定序 (next-generation sequencing，NGS) 7 5. Gene ontology (GO) term 分析 7 6. 引子對設計 7 7. RNA萃取 8 8. 反轉錄聚合酶鏈式反應 (RT-PCR) 8 9. 聚合酶鏈式反應 Polymerase chain reaction 8 10. 即時聚合酶鏈式反應 Real-time polymerase chain reaction (RT-qPCR) 9 11. 發芽率 (germination rate) 測定 9 12. 存活率 (survival rate) 測定 9 13. 根延長 (root elongation) 測定 10 14. 細胞色素含量測定 10 15. 抗氧化酵素 (APX、CAT) 萃取 10 16. 抗氧化酵素POX萃取 11 17. 抗氧化酵素SOD萃取 12 18. H2O2含量測定 12 19. 丙二醛 (MDA) 含量測定 12 20. DNA 萃取 13 21. DNA保護測定 13 結果 14 1. 確認轉基因菸草之IbTLD基因表現 14 2. 內源性菸草之NtTLD基因表現 14 3. 鹽逆境對於菸草外表型的影響 14 4. RNA sequencing中基因表現篩選與Gene ontology-term分析 15 5. 轉基因菸草中逆境反應相關基因的表現量分析 16 6. 抗氧化酵素活性 17 7. 鹽逆境對菸草植株內MDA以及H2O2含量的影響 17 8. IbTLD對DNA保護的影響 18 討論 19 1. 鹽逆境下TLDc與種子萌芽、存活率、根長以及細胞色素含量的關係 19 2. TLDc與氧化逆境所誘導的抗氧化逆境基因分析 19 3. TLDc、鹽逆境與H2O2以及MDA的關係 21 結論 23 圖表 24 表一、本論文所使用引子 24 表二、受到誘導而表現量上升基因 25 表三、其他物種之TLDc 26 圖一、IbTLD轉錄表現量分析 27 圖二、NtTLD轉錄表現量分析 28 圖三、鹽逆境對種子發芽率的影響 29 圖四、鹽逆境對存活率的影響 30 圖五、鹽逆境對根長的影響 31 圖六、鹽逆境對葉綠素與類胡蘿蔔素含量的影響 32 圖七、RNA-sequencing以及Gene Ontology-term網路分析 33 圖八、逆境反應基因轉錄表現量分析 34 圖九、鹽逆境對抗氧化酵素活性的影響 35 圖十、鹽逆境對H2O2含量的影響 36 圖十一、鹽逆境對丙二醛 (MDA) 含量的影響 37 圖十二、逆境反應基因轉錄表現量分析 38 圖十三、轉殖株中因鹽而導致之DNA ladder檢測 39 圖十四、IbTLD在遭受氧化逆境與鹽逆境時的調控反應 40 參考文獻 4

Effect of NaCl on the growth and development of rice seedling roots

本論文係以水稻台中在來一號 (Oryza sativa L. cv. Taichung Native 1) 為材料，探討鹽分逆境下，Ca2+、nitric oxide (NO) 及auxin 對水稻黃化幼苗主根生長及冠根形成之影響。 高鹽逆境 (150 mM NaCl) 會抑制水稻冠根之形成，而處理氯化鈣 (CaCl2)、一氧化氮釋放劑 sodium nitroprusside (SNP) 及植物生長素indole -3- acetic acid (IAA) 可顯著減緩高鹽逆境對冠根形成的抑制，進一步利用石蠟切片發現冠根原基發育與上述結果一致。而處理一氧化氮清除劑2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl -3-oxide (cPTIO) 結果則抑制CaCl2的作用，顯示高鹽逆境下NO參與Ca2+ 調控冠根發育之機制。利用 nitrate reductase (NR) 抑制劑sodium tungstate處理後可完全抑制高鹽逆境下Ca2+ 恢復冠根發育的作用，但 nitric oxide synthase (NOS) 抑制劑Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) 處理則無顯著影響，結果顯示高鹽逆境下 Ca2+ 主要經由NR途徑生成NO。以auxin 極性運移influx蛋白抑制劑1-Naphthoxyacetic acid (1-NOA) 及efflux蛋白抑制劑N-1-Naphthylphthalamidic acid (NPA) 處理後，Ca2+ 及NO之作用僅受NPA抑制，推測IAA極性運移也參與Ca2+ 及NO恢復水稻幼苗於高鹽逆境下冠根形成之作用。且高鹽逆境下CaCl2 恢復冠根之形成不受guanylate cyclase (GC) 抑制劑 LY83583 及 calmodulin 拮抗劑 chlorpromazine hydrochloride (CPZ) 影響。 低鹽逆境 (25 mM NaCl) 可誘導水稻冠根之形成，但可被1-NOA、NPA、cPTIO 及L-NAME處理所抑制，且 IAA 能明顯恢復 cPTIO 之抑制效果。CPZ、cyclic ADP ribose (cADPR) 合成抑制劑 NA、Ca2+ 螯合劑 EGTA、Ca2+ 原生質膜通道抑制劑 LaCl3 與 ruthenium red (RR) 處理皆不影響低鹽逆境誘導之冠根形成，但受LY83583、inositol 1,4,5-trisphosphate (IP3) 循環抑制劑 LiCl 所抑制。推測低鹽逆境下可能是經IP3 調控細胞質 Ca2+ 濃度，影響 NO、IAA 及cGMP 進而誘導冠根之形成。 處理CaCl2 可明顯減緩高鹽逆境抑制之主根生長，且CaCl2 之作用可被CPZ所抑制，顯示高鹽逆境下 CaCl2 可能經由 calmodulin 減緩主根生長受抑制之情形。In this thesis, rice (Oryza sativa L. cv. Taichung Native 1) seedlings were used to investigate the role of Ca2+, nitric oxide (NO) and auxin in the regulation of primary root (PR) growth and crown root (CR) emergence in salt stress. In high salt stress (150 mM NaCl), the CR of rice seedlings was reduced. Application of calcium chloride (CaCl2), sodium nitroprusside (SNP; a NO donor) and indole-3-acetic acid (IAA) to rice seedlings induced CR emergence in high salt stress. Further research on the CR primordia by paraffin section, the result is consistent with above. The effect is specific for NO because the NO scavenger 2-(4-carboxyphenyl)- 4,4,5,5-tetra-methylimidazoline-1-oxyl-3-oxide (cPTIO) blocked the action of CaCl2. These results suggest the involvement of NO in regulating Ca2+ induced CR emergence in high salt stress. Nitrate reductase (NR) inhibitor sodium tungstate inhibited Ca2+ induced CR emergence in high salt stress. In contrast, nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) was not significant. It means that NO generation that occurs in response to Ca2+ might primarily involve NR activity in high salt stress. Treatment of seedlings with auxin polar transport influx protein inhibitors 1-Naphthoxyacetic acid (1-NOA) and efflux protein inhibitors N-1-Naphthylphthalamidic acid (NPA), the result indicate that only NPA reduced to response in both Ca2+ and NO. Moreover, guanylate cyclase (GC) inhibitor LY83583 and calmodulin antagonists (CPZ) had no effect on Ca2+ induced CR emergence in high salt stress. On the other hand, low salt stress (25 mM NaCl) increased the rice seedlings CR emergence, which could be blocked by 1-NOA, NPA, cPTIO and L-NAME. Moreover, application of rice seedlings with IAA resulted in a significant increase in cPTIO treatment. Supplemental CPZ, cADPR synthesis inhibitor (NA), Ca2+ chelator (EGTA), plasma membrane Ca2+-channels inhibitors (LaCl3, RR) had no effect on increased of CR emergence by low salt stress. However, low salt stress increased CR emergence could be reduced by LY83583 and IP3 cycling inhibitor (LiCl). Therefore, our results demonstrated that NO, IAA and cGMP are involved in Ca2+ induced the rice CR emergence in low salt stress. Moreover, cytosolic levels of Ca2+ maybe regulated by IP3. Treatment of CaCl2 to rice seedlings also induced PR growth in high salt stress, which could be reduced by CPZ. These results indicate that calmodulin are involved in Ca2+ induced the PR growth in high salt stress.中文摘要………………………………………………………..i 英文摘要………………………………………………………iii 目錄…………………………………………………………….v 圖表目錄………………………………………………………vi 縮寫字對照表………………………………………………..viii 前言…………………………………………………………….1 前人研究……………………………………………………….3 材料與方法…………………………………………………...17 結果…………………………………………………………...24 討論…………………………………………………………...57 未來研究方向………………………………………………...66 參考文獻……………………………………………………...6

Quantitative trait loci mapping for rice root traits under salinity stress at seedling stage

　　水稻是世界上重要糧食作物之一，隨著人口增長，產量勢必要向上提昇。土壤鹽害是常見限制作物產量的非生物逆境，水稻在作物中屬於鹽敏感的作物，因此維持水稻在鹽逆境下的生長勢是日趨重要的議題。水稻面對鹽逆境時，根部首當其衝，影響著水稻對鹽分與養分吸收。本研究以 130 個秈稉雜交所產生的重組自交系族群及 196 個水稻種原為材料，調查幼苗期種子根、種子根上側根、冠根以及總根長在鹽逆境下的表現，發現根長性狀受鹽逆境抑制生長的程度不同；另外調查幼苗株高、鹽害指數等地上部性狀在鹽逆境中的反應，發現根部在鹽逆境長度愈大，通常幼苗株高也愈大。接著利用高密度單一核苷酸多型性分子標幟 (single nucleotide polymorphism, SNP) 進行單點分析 (single marker analysis) 並定位調控水稻耐鹽能力的數量性狀基因座 (quantitative trait locus, QTL)。本研究在兩族群中共偵測到 6 個基因座可能影響鹽害指數，我們分析鹽害指數表現優良的品系在這些基因座中的基因型，發現這些基因座偏屬japonica 次族群。另外 地上部性狀與根部性狀具有正相關性，但是並沒有偵測到相同的基因座；本研究亦定位到數個數量基因座可能影響根長性狀在鹽逆境的變異程度，在染色體 1、4、10 號上定位到重疊的基因座控制不同型態的根生長。鹽逆境中根部性狀表現優良的品系可以做為育種材料，經由分子標幟輔助育種將定位到的基因座導入現行品種，改良根部性狀在鹽逆境的生長勢。　　Rice is one of the major crops in the world. Because human population grows, increasing rice production is necessary to meet human’s need. Salinity is a common abiotic stress to affect crop production, and rice is one of salt sensitive crops, therefore maintaining rice growth vigor under salt condition is an important issue. Root system is a major physical interaction between plants and soil substrates, it affects nutrients and salt absorption. In this research, we used two populations as study materials, one is recombinant inbred lines population derived from a cross between indica and temperate japonica, and a global diversity panel with 196 accessions. We measured root length under normal and salt condition at seedling stage, and found out degree of root growth vigor inhibited by salinity stress differs by root types. We used high density SNP markers to map QTLs controlling shoot and root growth in response to salt stress, many QTLs in RILs and in global diversity panel were mapped in this study. Although there was positive correlation between root and shoot traits under salinity stress, no identical QTLs were mapped. We selected accessions with good performance under salinity stress, these accessions will be good breeding materials to improve root vigor in elite cultivars, and through molecular assisted selection, QTLs with positive effect can be introduced to elite cultivars efficiently.誌謝…………………………………………………………………………………....…i 中文摘要……………………………………………………………………………...…ii 英文摘要……………………………………………………………………………......iii 目錄……………………………………………………………………………...……...iv 表目錄……………………………………………………………………...……...……vi 圖目錄……………………………………………………………………….……...…viii 附件目錄………………………………………………………………………….......…x 簡寫對照表…………………………………………………………………….…….....xi 一、前言…………………………………………………………………………..……1 二、前人研究………………………………………………………………………..…3 1. 鹽逆境對植物生長影響…………………………………………………..…...3 2. 鹽逆境對水稻的影響………………………………………………..………...3 3. 調控鹽害逆境的水稻基因座定位……………………………………...….….4 4. 水稻根系構造……………………………………………………...…………..6 5. 耐鹽機制…………………………………………………………...……….….8 6. 水稻幼苗期生長勢………………………………………………...…….…….9 7. 關聯性定位法………………………………………………………...….…….9 三、材料與方法………………………………………………………………….……11 1. 試驗材料…………………………………………………………………..…..11 2. 實驗流程與設備使用…………………………………………………………12 3. 數量性狀基因座定位……………………………………………………..…..19 4. 統計軟體使用……………………………………………………………..…..24 四、結果………………………………………………………………………………..26 1. 實驗條件決定……………………………………………………………..…..26 2. 材料族群結構與親緣關係………………………………………………..…..26 3. 外表型調查……………………………………………………………………27 4. 數量性狀基因座定位…………………………………………………..……..35 5. 以重組自交系族群 SNP 建立連鎖圖譜並定位根部數量性狀基因座.........39 五、討論………………………………………………………………………………..41 1. 軟體分析根部數據……………………………………………………..……..41 2. 外表型變異……………………………………………………………..……..41 3. 鹽害指數表現優良品系………………………………………………..……..41 4. 性狀間相關性比較………………………………………………………..…..42 5. 綜合正常水耕環境測量值篩選鹽逆境中的表現優異的品系…………....…45 6. 兩族群數量性狀基因座定位結果比較………………………………………46 7. Sliding window size 造成連鎖圖譜的差異………………………………..…47 六、結論……………………………………………………………………………….49 表…………………………………………………………………………………...…..51 圖……………………………………………………………………………………….81 七、參考文獻………………………………………………………………………….166 附錄………………………………………………………………………………...…17

Functional Studies of FIN219 Involved in Drought and Salt Stress in Arabidopsis thaliana

當植物面臨到生長的逆境時，會藉著賀爾蒙調節生長發育。茉莉酸和乙烯便是目前發現參與多種生物逆境以及非生物逆境的荷爾蒙。FIN219 (FAR-RED INSENSITIVE 219)是一個已被報導參與在茉莉酸生合成的酵素，FIN219可以協助茉莉酸和異白氨酸結合，而接上異白氨酸的茉莉酸為具有活性的茉莉酸，可調節植物的生長及發育。除此之外，FIN219也參與在光訊息傳遞，實驗室前人的研究發現，FIN219可和藍光的接受器隱花色素中的CRY1（cryptochrome 1）在藍光中有互相拮抗的作用。由於目前學界發表的論文中，提到隱花色素的雙突變植株cry1/2對於乾旱具有耐受性，因此FIN219和CRY1之間的拮抗作用暗示FIN219在鹽逆境及乾旱逆境參與的可能。除FIN219和CRY1的關係外，有更多證據顯示FIN219可能會參與在鹽逆境以及乾旱逆境，包括實驗室先前對於FIN219和乙烯之間的交互關係的研究，以及微陣列分析數據顯示FIN219會影響到多個ERF (Ethylene Responsive Factor)，都顯示FIN219參與在鹽逆境以及乾旱逆境的可能性。目前我的研究結果顯示，FIN219在鹽逆境的處理之下會改變在細胞中的分布情形，而在特定的鹽逆境處理之下，FIN219會有較低的存活率。在乾旱逆境中，fin219-2 的植株的乾重則會明顯的比野生型的植株重。real-time PCR的結果顯示在鹽逆境之下，fin219-2會影響多個ERF 的表現量。另外在Pull-down assays顯示受FIN219調節的HY5 (Long hypocotyl 5)會和幾個ERF結合。且經過初步的根長測試，erf9和erf13突變體在鹽逆境下的根長受到明顯的抑制。綜合以上所述，推測FIN219可能藉由與CRY1 以及HY5參與在鹽逆境及乾旱逆境之中，我們將進一步的檢驗ERF在鹽逆境及乾旱逆境的表現，來釐清FIN219在這些逆境之中參與的分子機制。Being immobile, plants use hormones to control their growth when they encounter abiotic stresses. Jasmonates, mainly associated with biotic stresses, also participate in abiotic stress responses. FIN219 (FAR-RED INSENSITIVE 219) is a protein recognized in light signaling pathway, and also discovered to have the ability to conjugate jasmonic acid (JA) with isoleucine (Ile), leading to the formation of JA-Ile. Our lab found that FIN219 and one of blue light receptors-CRY1 (cryptochrome1) antagonized with each other under blue light condition. Previous study revealed that the cry1/2 mutant showed a drought resistance. FIN219 was able to inhibit the CRY1 levels, which suggests the involvement of FIN219 in salt and drought stresses. Moreover, our previous studies indicated that FIN219 played a role in ethylene response and affected several ERF (ethylene responsive factor) transcription factors. Current data showed that FIN219 changed its location under salt stress and the survival rate of fin219-2 seedlings was also reduced under salt stress. However, the growth of fin219-2 was less inhibited compared to Col-0 under osmotic stress. Moreover, FIN219 protein levels decreased under salt and drought stresses. These data indicate that FIN219 may participate in drought and salt responses. Quantitative real-time PCR analyses revealed that some ERFs expressed differently under salt stress between Col-0 and fin219-2. Besides, the levels of HY5 (ELONGATED HYPOCOTYL 5) changed under both stresses. Pull-down assays showed the interaction between HY5 and several ERFs. Further studies revealed that the root lengths of erf9 and erf13 were influenced under salt stress, indicating the involvement of ERF9 and ERF13 in salt stress response. Taken together, the involvement of FIN219 in drought and salt responses may be through negatively and positively regulating cry1 and HY5-ERFs signaling networks. We will further examine the phenotype of ERF mutants under drought and salt stresses in order to confirm the involvement of FIN219 in these stress responses

Analysis of K+/Na+ ion homeostasis and related gene expression in NaCl stress environments.

當植物受到鹽逆境時，會因為細胞內累積過多的氯化鈉離子，而抑制細胞質酵素活性並造成細胞內外離子的不平衡，影響鉀離子的吸收，進而引發缺鉀及缺水等傷害。為探討植物細胞對鹽逆境下對鉀離子吸收之影響，本篇論文以耐鹽植物冰花(Mesembryanthemum crystallinum L.)及不耐鹽植物阿拉伯芥(Arabidopsis thaliana)之培養細胞為材料，施予鹽逆境並配合不同濃度之鉀離子處理，測量細胞生長狀況及細胞內鉀鈉離子的含量及比值，以評估不同植物間對於鹽逆境下離子平衡的差異性。由細胞在不同鈉鉀離子濃度的培養基中生長的狀況可以發現，雖然冰花細胞所能承受的鹽逆境高於阿拉伯芥細胞，但無論是冰花或是阿拉伯芥細胞，培養在低鉀的環境中會使對鹽逆境的敏感度上升，造成過高的鈉鉀比值。當細胞培養在以氯化鉀造成高鉀環境的培養基時，細胞生長會受到抑制，以非滲透性溶質mannitol或硫酸鉀取代氯化鉀之培養基，會改善生長抑制的情況，推測過高濃度之氯離子會抑制細胞生長。雖然高鉀環境會限制細胞之生長，但是冰花及阿拉伯芥細胞在高鉀高鹽培養初期，都可以藉由培養基所提供的高濃度鉀離子維持較低之鈉鉀比值。 除了評估冰花細胞生長及鈉鉀離子含量外，並利用一鉀離子運輸相關mcSKD1基因，觀察基因表現及細胞離子平衡的關係。由北方墨點法分析結果得知，mcSKD1基因在鹽逆境及缺鉀逆境初期表現量會下降，之後表現量會增加。配合之前的生理研究結果發現，鹽逆境下mcSKD1基因表現，可降低冰花培養細胞中鈉鉀比值，推測此基因參與鈉鉀離子平衡之機制。由mcSKD1基因在組織專一性的表現得知，在未受鹽逆境的環境下，mcSKD1在發育中的子房及根尖的表現，推測mcSKD1基因參與提供新生組織營養的功能。鹽逆境下mcSKD1基因會在根部的表皮、莖部的皮層以及葉部腎型細胞表現，與區隔鈉離子的作用相關；由冰花葉片的鉀鈉元素分析結果得知，當mcSKD1 基因在根部表現時，可提高葉片中鉀離子的含量，表現量不足時，葉片中鉀離子量逐漸下降，推測mcSKD1基因參與冰花耐鹽機制中維持細胞鉀離子平衡的途徑。When plants under high salinity stress, excess NaCl ions accumulate inside the cells, and as the results, the activities of cytosolic enzymes are inhibited and ion homeostasis is interfered. Moreover, the uptake of essential element potassium is severely affected. To examine the effects of high salt on the uptake of potassium, we used well-defined culture media to culture suspension cells of halophyte Mesembryanthemum crystallinum (ice plant) and glycophyte Arabidopsis thaliana. The growth rates and the contents of Na+ and K+ were measured in cells grown in different combinations of Na+ and K+ concentrations. Although ice plant had higher ability to grow in salt-containing media, yet the sensitivity to high salt increased as the concentration of K+ decreased in the culture media in both plant cells. High KCl-containing media inhibited the growth rate of both cells and replacement of KCl by mannitol or K2SO4 would alleviate the inhibitory effect suggesting that high concentration of Cl- in the culture media caused this growth inhibition. At the initial stage of salt stress, both cells maintained lower sodium/potassium ratios when a high external K+ was supplied. In addition to the growth and ion content measurements, the expressions of an ice plant gene mcSKD1 that has been related to potassium uptake were also examined. As the results from Northern blotting, the expression of mcSKD1 decreased at the initial stage of high salt or low potassium treatments. The levels of mcSKD1 transcript increased as the stress persisted. The increase of mcSKD1 expression parallels to the decrease of cellular Na+/K+ ratio suggesting that this gene be involved in the regulation of ionic balance in ice plant under salt stress. Tissue-specific expression found mcSKD1 gene was expressed in the developing ovary and root tips of the unstressed plants suggesting this gene is involved in nourishing the young developing tissues. Under salt stress, the expressions of mcSKD1 were found in tissues that are responsible for the compartmentation of excess Na+, such as the epidermal cells of roots, the cortex of stems and the specialized epidermal bladder cells of leaves. Together with the analyses of sodium and potassium ion contents, we showed the expression of mcSKD1 in the roots was parallel to the increase of potassium concentrations in the leaves. The function of mcSKD1 protein may play a part in maintaining the potassium homeostasis in salt-stressed ice plant, an important pathway contributing to the overall mechanism of salt adaptation in this halophyte.中文摘要………………………………………………………..…………………I 英文摘要………………………………………………………….………………II 壹、 前言：………………………………………………………………………. 1 一、 耐鹽植物冰花……………………………………………………..2 二、 鹽逆境下阿拉伯芥的反應………………………………………..3 三、 鹽逆境下鉀離子在植物細胞內離子平衡上所扮演的角色……..4 四、 冰花耐鹽相關基因mcSKD1………….…………………………..6 貳、 材料與方法:………………………………………………………………….8 一、 實驗材料：…………………………………………………………8 （一） 冰花盆栽……………………………………………………..8 （二） 細胞培養……………………………………………………..8 二、 實驗方法：………………………………………………………..9 （一） 植物體中Na, K, Ca, Mg之測定…………………………….9 （二） Total RNA之萃取…………………………………………..10 （三） 北方墨點法分析……………………………………………11 （四） 利用RT-PCR方法擴增全長SKD1基因…………………..12 （五） PCR產物之選殖……………………………………………13 （六） In situ RT-PCR and In situ hybridization…………………...14 （七） 掃瞄式電子顯微鏡觀察冰花葉表腎型細胞………………18 參、 結果…………………………………………………………………20 肆、 討論……………………………………………………………………29 伍、 參考文獻………………………………………………………………3

Functional analysis of Arabidopsis WRKY63 in salt stress response and flowering time control

WRKY轉錄因子在植物界中是一個很大的蛋白家族。在先前的研究當中，阿拉伯芥WRKY轉錄因子被報導在生物性防禦反應中扮演十分重要的角色，但在非生物逆境與植物生長發育的調控中的作用所知仍然較少。在本研究中我們發現屬於IIIa類的WRKY轉錄因子WRKY63蛋白會和RPD3類的組蛋白去乙酰化酶HDA6有交互作用，我們也觀察到WRKY63的突變株abo3對鹽逆境及離層酸有敏感的表型，並且發現WRKY63和HDA6的雙突變株abo3/axe1-5對鹽逆境會有較單突變更敏感的表型；另外，我們也觀察到abo3突變株有比野生型早開花的表型。透過檢測這些突變株與野生型植株中的基因表達，我們發現鹽逆境反應與開花調控的下游基因表達，相對於野生型在突變株中皆有受到影響。從這些結果我們可以知道WRKY63可能會藉由調控下游基因的表達來影響鹽逆境反應及開花時間的控制。WRKY transcription factors constitute a large protein family in plants. Previous studies indicated that WRKY transcription factors play important roles in the regulation of gene expression associated with plant defense responses, but the role of WRKY proteins in abiotic stress responses and development is still unclear. In this study, we show that WRKY63, a member of WRKY IIIa subfamily, could interact with the RPD3-like histone deacetylase HDA6. The WRKY63 T-DNA insertion knock-out mutant, abo3, is hypersensitive to ABA and salt stress. Furthermore, abo3/axe1-5 double mutant plants displayed increased salt sensitivity compared with abo3 and axe1-5 plants. In addition, the abo3 mutant showed the early flowering phenotype compared with wild type plants. The expression levels of salt stress-responsive genes as well as genes involved in flowering time control were changed in the abo3 mutants. These data suggested that WRKY63 plays an important role in the regulation of gene expression involved in salt stress responses and flowering time control

The Rice Transcription Factor, OsbHLH046, Contributes to Salt Stress Tolerance at Seedling Stage

水稻bHLH轉錄因子(basic helix-loop-helix transcription factor, bHLH TF)為重要之轉錄因子多基因家族，參與諸多生理功能，包括生長發育以及對抗非生物逆境等。由於轉錄因子多基因家族功能互補特性，如何確認特定轉錄因子之功能在研究上有一定之困難度。在本篇研究中，我們由實驗室先前水稻DNA微陣列的分析結果找出了在鹽及乾旱逆境下基因表現受抑制之轉錄因子OsbHLH046，並進一步以RT-PCR確認之。根據OsbHLH046-GFP之融合蛋白表現分析，發現OsbHLH046為一核蛋白。而Rice eFP browser與OsbHLH046 pro::GUS轉植株染色結果分析則顯示OsbHLH046在水稻胚、種子根、側根、幼苗時期地上部與花藥等部位皆有表現，其表現隨著幼苗葉齡漸大而有減弱之趨勢，但於成熟之花藥仍具高表現量。而OsbHLH046之Knock-down Tos17插入突變株相比於野生型植株有著較差的發芽率，較低的株高與較差的鹽逆境恢復性。丙二醛之含量測定亦顯示Osbhlh046突變株於鹽逆境下含量較野生型植株高。另外，qRT-PCR結果顯示部分鹽逆境耐受相關基因如OsNHX2與OsABI5在鹽逆境恢復階段中於Osbhlh046與野生型植株彼此間有著差異表現。而在ICP-OES元素分析的結果中，發現Osbhlh046植株在地下部於鹽逆境處理後復水五天，其K+/Na+比值較高，相較於野生型，無法維持Na+之恆定性而恢復性較差。最後，OsbHLH046過量表現系之癒傷組織相比於其他轉殖系，再生率差值達74%左右。綜上所述， OsbHLH046應參與種子發芽、植株生長，並且於鹽逆境下作為ㄧ正向調控因子參與水稻之耐受性。Rice basic-Helix-Loop-Helix (OsbHLH) transcription factors (TFs) belong to a multiple gene family and are known to involve in rice growth, development, regulating abiotic stress-responsive gene expression and tolerance of rice. However, because functional redundancy within the super gene family and the pleiotropic effect of individual TF, the specific function for corresponding OsbHLH is not easy to be determined. In this study, we identified an OsbHLH046 TF which is down-regulated by salt and cold treatments from previous microarray result and further confirmed the gene expression by reverse transcription-PCR (RT-PCR). The OsbHLH046-GFP fusion protein showed OsbHLH046 is located in nucleus. Based on the predication of rice eFP browser and the GUS-histochemical staining indicated that OsbHLH046 is expressed in the embryo, seedling root, lateral roots, shoots and anthers. The gene expression was higher at the early germination stage then declined to lower level within 3rd leaf stage. Compared with WT, the Osbhlh046, a Tos17 knocked-down rice mutant, displayed late seed germination, growth retardation, and unable to recover from salt stress at seedling stage. The q-PCR data indicated that salt tolerance related genes in such as OsABI5, OsNHX2 were differential expressed between two plants during recovery stage. The MDA was overaccumulated in Osbhlh046 mutant compared to wild type after recover from salt stress. The ICP-OES elemental analysis also revealed that the Osbhlh046 plant have lower K+ content level, which may not able to maintain the Na+ homeostasis. Meanwhile, the callus of OsbHLH046 overexpression line showed a relative poor regeneration rate. Taken together, these results suggested that OsbHLH046 plays as a positive regulator in seed germination, seedling growth, and contributes to the ability of rice to recover from salt stress

Establishment of optimal growth condition and the effects of exogenous myo-inositol on salt tolerance in seedlings of ice plant (Mesembryanthemum crystallinum L.)

土壤環境中鹽類含量過高會導致植物處於鹽逆境與滲透逆境，甚至進一步產生氧化逆境。肌醇(inositol)為一相容質，於水份含量相關之鹽、乾旱及低溫逆境下會大量累積於細胞質以協助植物適應逆境，此外肌醇衍生物也參與植物適應逆境的過程，例如pinitol、galactinol、raffinose與ascorbate等，除了具有相容質之功能外，也具有清除ROS (reactive oxygen species)之能力。文獻中指出，耐鹽模式植物冰花(Mesembryanthemum crystallinum)於鹽逆境下可藉由肌醇運輸蛋白(inositol transporter; INT) McINT4.1/MITR1與McINT4.2/MITR2使鈉離子於植物體內重新分佈，以降低根部鈉離子累積，為冰花適應鹽逆境的重要機制之一。經由冰花轉錄體搜尋出7個INT基因，以親緣關係和序列相似性依照阿拉伯芥INT家族重新命名為：McINT1.1、McINT1.2、McINT2、McINT3、McINT4.1、McINT4.2與McINT4.3。本論文主要探討外源性肌醇對鹽逆境下冰花小苗生長、鈉鉀離子含量及肌醇運輸機制以及McINT家族表現量差異。依據本實驗室先前培養無菌冰花小苗的條件，冰花小苗容易有玻璃質化的現象，離開無菌環境後容易脫水造成實驗誤差，故本論文測試了培養基的組成與培養條件，發現藉由通氣可降低小苗玻璃質化的現象，成功獲得健康且均質化的小苗進行後續實驗。此外，本實驗室先前處理冰花小苗的方式有小苗處理不完全與使小苗處於淹水逆境之疑慮，而本論文也改以直立浸泡的方式解決以上問題，並且可以使同一培養皿內之冰花小苗同時進行多種處理，以降低可能因培養於不同培養皿所造成的誤差。本論文觀察到鹽逆境下添加外源性肌醇可以減緩冰花小苗脫水的現象，而檢測McINT表現量，發現於不同溶液處理下，地上部與地下部McINT有不同的表現趨勢。地上部所有McINT皆受到鹽逆境而表現量上升，其中地上部McINT3、McINT4.2與McINT4.3受到肌醇負調控有表現量下降的趨勢，而於地下部，鹽逆境下無論有無外源性肌醇，McINT皆有表現量上升的趨勢，且沒有任何McINT受到肌醇負調控，顯示不同群的McINT有特定的功能與調控機制。於追蹤外源性肌醇與分析鈉離子累積之實驗中發現，冰花小苗具有吸收環境中微量肌醇的能力，且鈉離子可以促進外源性肌醇之吸收，於長時間鹽逆境下，添加外源性肌醇可以降低根部鈉鉀比值。植物於鹽逆境下會導致氧化逆境的發生，而本論文發現冰花小苗於各處理下其ROS的累積程度並沒有明顯的差異，此結果可能因背景值過高而需要進一步進行定量分析。綜合上述，本論文初步鑑定了冰花小苗McINTs之功能及參與調控機制，冰花小苗可藉由增加基因表現或蛋白活性促進鈉離子累積於地上部並降低地下部的鈉鉀比值，以增加小苗對鹽逆境的耐受性。Plants develop many mechanisms to adapt to the change of environment, but if the environment becomes more extreme, plants fail to respond and result in the stresses. Stresses can be distinguished into two classes, one is the biotic stress that is involved in interaction with other organisms, and the other is the abiotic stress caused by the change of the environmental factors. Excessive salt content in the soil environment will cause plants encountering salt, osmotic and even oxidative stress. Inositol is a compatible solute that accumulates in the cytosol and organelles to facilitate plant to adapt to water-deficit related stresses. Inositol derivatives such as pinitol, galactinol, raffinose and ascorbate are also involved in the processes of stress adaptation. These derivatives are also compatible solutes and have the ability to scavenge the ROS (reactive oxygen species). Halophyte ice plant (Mesembryanthemum crystallinum) is a model organism to study plant salt tolerance. Ice plant can redistribute the sodium ions in the plant under the salt stress via inositol transporter (INT) McINT4.1/MITR1 and McINT4.2/MITR2. It is one of the vital mechanisms for ice plant to adapt to salt stress. We identified seven INT genes form ice plant transcriptome and, according to the classification of Arabidopsis thaliana AtINT family, renamed as McINT1.1, McINT1.2, McINT2, McINT3, McINT4.1, McINT4.2 and McINT4.3. According to the previous culture condition of the ice plant seedlings, the seedlings tended to develop vitrification in the sealed condition. To solve the problem, we modified the compositions of the culture medium and culture conditions and successfully obtained healthy and uniform seedlings. Furthermore, the previous method of seedling treatment has the doubts about the incomplete treatment of roots and seedlings were under flooding stress. Therefore, a vertical immersion of seedlings was used to avoid the problem described. I found that the supply of exogenous myo-inositol can reduce the dehydration effects of salt-stressed seedlings. Differential expression analysis of McINTs showed that all McINTs were induced by salt stress at the shoot, and the expressions of McINT3, McINT4.2 and McINT4.3 were down-regulated by inositol. In root, the expression of all McINTs also induced under salt stress with or without exogenous inositol supply, and none of McINTs was down-regulated by inositol. This result indicated that different McINTs might have different functions and regulation in ice plant seedlings. Analyses of the uptake of exogenous inositol and accumulation of sodium ions revealed that ice plant seedlings had intrinsic ability to take up a trace amount of external inositol and the presence of sodium could facilitate inositol uptake. Under prolong salt stress, an exogenous supply of inositol decreased the Na/K ratio in roots. Oxidative stress is usually induced when plant suffer in salt stress. Ice plant seedlings have no significant differences in ROS accumulation by salt treatments. Quantification of ROS is needed for further confirmation. In conclusion, I identified the possible functions and regulatory mechanisms of ice plant McINT. Ice plant seedlings can accumulate more sodium in shoot and decrease the Na/K ratio in the root via induced specific McINT gene expression or activated transport activity of specific member of McINT to enhance the tolerance of salt stress.摘要 i Abstract ii 目次 iv 表目次 vi 圖目次 vii 壹、前言 1 一、鹽逆境對植物生長之影響 1 二、植物適應鹽逆境之機制 2 (一) 離子運輸與儲存 2 (二) 小分子相容質之累積 3 (三) 清除活性氧之途徑 3 三、肌醇在植物對抗逆境之角色位置 4 (一) 肌醇生合成與甲基化的肌醇 5 (二) 活性氧清除分子 6 (三) 肌醇運輸蛋白 7 四、模式生物冰花 9 五、研究目的 10 貳、材料與方法 11 一、實驗材料 11 (一) 冰花(Mesembryanthemum crystallinun)小苗無菌栽培 11 (二) 培養基與處理溶液配方 11 (三) 冰花小苗測試處理 11 二、實驗方法 12 (一) McINT之基因表現量分析 12 1. 冰花total RNA萃取 12 2. 反轉錄聚合酵素連鎖反應(Reverse transcription- polymerase chain reaction, RT-PCR) 13 3. 即時定量反轉錄聚合酵素連鎖反應(Quantitative reverse transcription polymerase chain reaction, qRT-PCR) 13 4. 洋菜膠體電泳分析 (DNA agarose electrophoresis) 14 (二) 檢測放射性肌醇[3H]myo inositol吸收與運輸 14 (三) 檢測冰花小苗植體內Na及K含量 15 1. 坩鍋前置處理 15 2. 樣品高溫灰化及強酸萃取 15 (四) 活性氧(reaction oxygen species, ROS)檢測 15 1. Nitroblue tetrazolium (NBT)染色分析 15 2. 3,3'-diaminobenzidine (DAB)染色分析 15 參、結果 17 一、冰花McINT序列比對與命名 17 二、無菌栽培冰花小苗最適生長條件 18 三、醣類分子影響冰花小苗McINT表現量 19 四、冰花小苗經鹽和myo-inositol處理後之外表型變化 19 五、冰花小苗鹽逆境下添加外源性肌醇對McINTs表現分析(RT-PCR) 20 六、冰花小苗鹽逆境下添加外源性肌醇對McINTs表現定量分析(qRT-PCR) 20 七、鈉離子運輸累積位置分析 21 八、外源性肌醇運輸累積位置分析 21 九、過氧化氫與超氧陰離子累積分佈位置 22 肆、討論 23 一、McINTs功能與調控機制之探討 23 二、鹽逆境下肌醇與Na分佈 24 三、外源性肌醇與ROS清除之探討 25 四、總結與未來展望 26 伍、參考文獻 27 陸、附錄 6

Characterization and Application of Osmolyte Synthetic Genes of Methanogenic Archaea

極端厭氧的甲烷太古生物 (Archaea, 又稱古菌、古生菌或古細菌)，以甲烷的生合成方式提供細胞所需的能量，能生存於各式溫度(2- 117 ℃)、酸鹼度(中性至強鹼)及鹽度(淡水至飽合鹽度)等環境，是唯一能適應多變化環境的太古生物。我們實驗室收集國內外能生長在各種不同鹽度的甲烷菌，長期探討甲烷菌對鹽逆境的反應與適應機制。我們的研究顯示嗜鹽甲烷菌具有一高親和性、高專一性，需能且受滲透壓調控的ABC型式的甜菜鹼運輸系統Bta，會自胞外環境優先攝取相容質甜菜鹼。而當胞外環境缺乏相容質的提供時，能自體生合成相容質β-glutamine、Nε-acetyl-β-lysine及甜菜鹼 (betaine)於細胞體內，以維持膨壓及保護胞內蛋白等大分子。除了利用各種特殊相容質的累積來適應環境中的不同鹽度之外，我們最近的研究首次發現分子伴蛋白chaperone ClpB亦參與鹽逆境的反應，在高鹽、低鹽逆境的表現量會增加，且會受相容質甜菜鹼的抑制。為探討相容質與甲烷古菌對鹽逆境的適應機制，我們目前已獲得嗜鹽甲烷菌Methanohalophilus portucalensis FDF1相容質betaine的生合成酵素GSMT與SDMT的基因。在甲烷菌的共通相容質Nε-acetyl-β-lysine生合成基因部分，我們已分別獲得海洋甲烷菌Methanosarcina mazei N2M9705、耐鹽甲烷菌Methanocalculus chunghsingensis K1F9705b與嗜鹽甲烷菌Methanohalophilus portucalensis FDF1的lysine 2,3-aminomutase (ablA)與β-lysine acetyltransferase (ablB)基因；以及淡水型甲烷菌M. mazei N2M9705的相容質β-glutamine 的生合成基因。在此三年期計畫期間，將分析甲烷菌相容質生合成基因的特性與系統演化分析；探討不同鹽度、溫度或相容質betaine 等因子對甲烷菌自體生合成的相容質基因之轉錄表現的影響與調控。此外，將相容質生合成相關基因分別轉殖到大腸桿菌大量表現並純化蛋白進行酵素活性分析；並將生合成相容質β-glutamine、Nε-acetyl-β-lysine及betaine的基因組，分別轉殖到大腸桿菌與阿拉伯芥上，分析其相容質生成累積量與探討是否能提高其對鹽度的耐受性。相容質與滲透壓的基礎與應用研究，從抗鹽相容質glycerol、trehalose和近年發展的高鹽細菌生產的ectoine在發酵工業、食品界、化妝美容界、製藥與醫療等的應用，可以想見微生物因應鹽與滲透壓力的小分子相容質在基礎研究與應用的重要性。絕對厭氧的甲烷太古生物能生長在淡水至飽和鹽環境，對鹽逆境與滲透壓逆境的適應是生物界中最寬廣的，期由對甲烷太古生物在鹽逆境的反應與適應機制相關基因與調控的瞭解及應用潛力的分析，進而提高生物抗鹽抗旱的能力

Comparative Analyses of Shoots and Roots Transcriptomics of Two Rice Seedlings (TNG67 vs. TCN1) under Cold or Salt Stress and Subsequent Recovery

摘要 近年來氣候變遷與環境變化所產生之逆境愈形劇烈，在各種非生物逆境下將導致作物品質與產量的水平下降，進而影響全球糧食的供給。隨著水稻基因組的解碼與功能性基因體技術的進步，專家學者們將能更進一步了解水稻抗逆境的機制並探索耐逆境相關的重要基因，以加速耐逆境水稻的育種過程。在此論文中，我們利用可耐低溫與鹽逆境之梗稻品種台農67號與對此兩種逆境敏感之秈稻品種台中在來1號為材料，並使用水稻表達譜晶片Rice OneArray® v1為轉錄分析平台進行研究。除了分析兩品種的莖部與根部於逆境下的基因表現，我們更進一步分析於逆境處理後之回復期的基因變化。結果顯示，台農67號於低溫下會促進檸檬酸循環(tricarboxylic acid)與程序性細胞凋亡 (programmed cell death)；於鹽逆境處理下會透過無氧呼吸(fermentation)產生能量並於鹽回復處理下提升卡爾文循環。此外，鹽逆境下Salt Overly Sensitive (SOS) 排鹽機制似乎對台農67號的耐鹽性有部分貢獻。根據與賀爾蒙相關之基因的表現趨勢進行推測，低溫下增強對離層酸(abscisic acid)、多元胺(polyamine)、茉莉花酸(jasmonic acid)與生長素(auxin)的反應會提升其低溫耐性。鹽逆境下，除了增加對離層酸、多元胺、茉莉花酸、生長素與乙烯(ethylene)的反應外，同時也需降低其對激勃素(gibberellin)及細胞分裂素(cytokinins)之反應，此結果顯示不同賀爾蒙之間的協同作用對耐鹽性之重要。而逆境消退後，乙烯與細胞分裂素的共同作用有利於水稻歷經低溫逆境後回復生長，而茉莉花酸則是參與在鹽逆境之回復期。在轉錄因子的調控方面，NAC與WRKY型之轉錄因子與低溫耐性具相關性，而MYB與AP2/ERF型之轉錄因子則可能參與耐鹽性機制。此外，兩品種於低溫與鹽逆境下所呈現出的“具差異性表現之基因”(differentially expressed genes)非常不同。雖然低溫與鹽逆境會導致類似的表徵性狀與生理損害，然而由其基因之表現可知，兩者於細胞層次上的分子調控機制存在很大的差異。若能愈清楚地了解水稻之低溫與鹽逆境耐性的機制，將有助於我們日後更精準的育成具不同非生物性逆境耐性的品種。Abstract Climate changes and environmental stresses become severe over the past few decades. In particular, different abiotic stresses reduce the yield and quality of crop, leading to the threaten of global food security. With the deciphering of rice genome and advancement of functional genomics technology, researchers were able to gradually reveal the mechanism of abiotic stress tolerance mechanisms in rice and to identify essential genes for breeding to improve stress tolerance. In this thesis, we used TNG67 (japonica) and TCN1 (indica) rice cultivars with contrastive tolerance to cold and salt stresses as studying materials. A custom designed oligonucleotide array, Rice OneArray® v1 microarray platform (Phalanx Biotech Group Inc.) was used for transcriptomic analysis of shoot and root tissues of these two cultivars under cold or salt treatment and subsequent recovery. The results showed that TNG67 which is tolerant to cold and salt stresses can enhance TCA (tricarboxylic acid) cycle and PCD (programmed cell death) pathways under cold stress while it shifts to fermentation pathway for energy production and enhances the efficiency of Calvin cycle under salt stress and recovery, respectively. In addition, activation of SOS pathway may partially contribute to salt tolerance of TNG67. Increase of genes expressions related to phytohormone biosynthesis and response of ABA, PA, JA, and auxin can help TNG67 in cold stress tolerance. Besides, maintaining the balance and crosstalk of different hormones through the induction of gene expressions related to ABA, ET, PA, auxin, JA and the decrease of gene expressions associated with GA and CK responses may also be quite important for salt tolerance of TNG67. The crosstalk of ET with CK and JA in rice may play a role in the restoration of cold and salt stress. Also, we investigated the possible transcription factors (TFs) which may be the candidate genes that control cold or salt stress tolerance in rice. The induction or repression of TFs under stresses includes NACs and WRKYs, and MYB and AP2/ERF. NACs and WRKYs were the major TFs that may participate in cold tolerance, and MYB and AP2/ERF may involve in salt stress tolerance. Taken together aforementioned results, the cold- and salt-tolerance exhibit distinct regulatory mechanisms in TNG67 vs. TCN1. Interestingly, comparing the DEGs in shoots or roots of both rice cultivars under stresses, the venn diagram analysis showed that TNG67 and TCN1 shared less differentially expressed genes (DEGs) between cold and salt treatment. Although cold and salt stress can cause similar phenotypes and physiological damages, the molecular basis of cellular regulation mechanism can be quite different. Understanding the difference of cold and salt tolerance mechanisms in details is important in the future for us to breed rice precisely to cope with various abiotic stresses