,, 王志城, 张新雨, 柯会锋, 吴立强, 王省芬, 张桂寅, 马峙英, 张艳,*华北作物改良与调控国家重点实验室 / 河北省棉花产业协同创新中心 / 河北农业大学, 河北保定 071001Function study of cotton GbSTK gene in regulating flowering and Verticillium wilt resistance
CUI Jing,, WANG Zhi-Cheng, ZHANG Xin-Yu, KE Hui-Feng, WU Li-Qiang, WANG Xing-Fen, ZHANG Gui-Yin, MA Zhi-Ying, ZHANG Yan,*State Key Laboratory of North China Crop Improvement and Regulation / Co-Innovation Center for Cotton Industry of Hebei Province / Hebei Agricultural University, Baoding 071001, Hebei, China通讯作者:
收稿日期:2020-04-11接受日期:2020-08-19网络出版日期:2021-01-12
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Received:2020-04-11Accepted:2020-08-19Online:2021-01-12
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崔静, 王志城, 张新雨, 柯会锋, 吴立强, 王省芬, 张桂寅, 马峙英, 张艳. 棉花GbSTK基因调控开花和黄萎病抗性的功能研究[J]. 作物学报, 2021, 47(1): 30-41. doi:10.3724/SP.J.1006.2021.04090
CUI Jing, WANG Zhi-Cheng, ZHANG Xin-Yu, KE Hui-Feng, WU Li-Qiang, WANG Xing-Fen, ZHANG Gui-Yin, MA Zhi-Ying, ZHANG Yan.
棉花(Gossypium spp.)是世界上重要的经济作物, 我国是棉花生产、消费、纺织品出口大国, 然而我国棉花生产面临黄萎病危害依然严重, 近年来新疆棉区发病面积迅速扩大, 危害日益加重, 因此对抗病品种的需求是生产上解决棉花抗黄萎病的根本。为了提升我国棉花在国际市场的竞争力, 棉花机械化的不断推进以促进植棉节本增效, 然而棉花播种覆盖的地膜成为机采过程中的严重白色污染, 造成棉花品质大幅下降, 为了从根本上缓解这一矛盾, 早熟棉的培育与应用显得尤为重要。因此, 抗病、早熟性研究是棉花要解决的重大生产和科学问题。
早熟性是作物重要的优良性状之一, 棉花的生育期包括营养生长(播种到现蕾)和生殖生长(开花到吐絮) 2个阶段。现有的棉花资源中发现早熟品种多抗性差、抗病品种多贪青晚熟; 并且植物本身也存在抗病-发育的调节平衡, 逆境条件下, 多数植株也会以暂停或减缓生长发育为代价而抵御逆境。建立抗病与早熟之间的联系、打破早熟与抗病的负相关、发掘同时控制开花与抗病的遗传因子对培育早熟抗病的品种具有重要意义。
蛋白磷酸化是真核生物中普遍存在的细胞调节机制[1]。丝氨酸/苏氨酸蛋白激酶(serine/threonine protein kinase, STK)是蛋白磷酸化过程中一类重要的蛋白因子[2], 在细胞信号传导中起着重要作用[3,4]。目前, STK在番茄[5,6]、水稻[7,8]、番木瓜[9]、拟南芥[10]、小麦[11,12]、烟草[13]、百合[14]等植物中均有发现, 研究表明STK参与植物抗病、干旱、光照等多个信号传导途径。本课题组前期鉴定了一个棉花丝氨酸/苏氨酸蛋白激酶基因(GbSTK), 该基因受黄萎病菌和多种信号分子诱导, 转GbSTK基因的拟南芥显著提高了植株黄萎病抗性, 有趣的是发现转基因拟南芥与对照相比, 开花期明显提前, 因此, 本研究进一步研究STK基因在棉花抗黄萎病和调控植株开花的功能, 以期为深入揭示棉花STK基因的分子机制奠定基础。
1 材料与方法
1.1 试验材料
转GbSTK基因拟南芥纯合株系[15]、黄萎病不同抗性棉花品种均由河北农业大学棉花遗传育种创新团队提供。强致病力黄萎病菌系LX2-1[16]由本实验室分离保存。EASYspin Plus植物RNA提取试剂盒购自北京艾德莱生物科技有限公司; PrimeScript 1st Strand cDNA Synthesis Kit反转录试剂盒、PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real-time)反转录试剂盒购自宝生物工程有限公司; 2×Phanta Master Mix购自南京诺唯赞生物科技有限公司; 限制性内切酶购自赛默飞世尔科技(中国)有限公司; T4 DNA连接酶购自普洛麦格公司; pMD19-T载体、大肠杆菌DH5α感受态细胞、质粒小提试剂盒、胶回收试剂盒购自天根生化科技(北京)有限公司; Matchmaker Gold Yeast Two-Hybrid System、诱饵表达载体pGBKT7、Aureobasidin A (AbA)、X-α-Gal购自Clontech公司; Y2HGold感受态细胞购自上海唯地生物技术有限公司。引物合成和测序由生工生物工程股份有限公司完成。1.2 黄萎病菌诱导下不同抗、感棉花品种STK基因的表达
采用水培法种植棉花不同抗、感品种, 每2 d更换1次Hoagland营养液。待棉苗二叶一心时, 将棉苗轻轻拿出, 将根部浸泡在黄萎病菌孢子悬浮液(1×107 spores mL-1)中3 min, 然后再将棉苗小心栽入上口直径6 cm的营养钵, 并从营养钵的底部再次注射10 mL的黄萎病菌孢子悬浮液, 以保证每棵棉苗成功接菌(附图1)。根据实验室前期构建的全长黄萎病菌诱导下cDNA文库中筛选的抗病相关基因的表达中发现, 参与防卫反应的基因一般在抗/耐病品种较感病品种的表达高峰出现早或表达水平高, 而前期我们对棉花STK受黄萎病菌诱导的荧光定量PCR显示, 在接菌后8~12 h, 其表达显著升高[15], 考虑到STK基因主要在参与信号转导方面起作用, 其发挥作用一般在接菌后的早期阶段, 因此本研究取接菌后12 h的根并提取其RNA, 用于研究多个抗感品种中STK基因的表达。根据GbSTK基因序列设计实时定量引物STK-F2和STK-R2 (表1), 片段大小为200 bp左右。根据PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real-time)反转录试剂盒合成cDNA。以棉花GhUBQ14[17]为内参基因(表1), qRT-PCR反应体系为10.0 μL, 包含1.0 μL cDNA、5.0 μL 2×SYBR、0.5 μL STK-F2 primer、0.5 μL STK-R2 primer、2.8 μL RNase-Free H2O、0.2 μL ROX校正液。反应程序为94℃预变性30 s; 95℃变性5 s, 57℃退火5 s, 72℃延伸34 s, 40个循环。试验设置3个生物学重复, 采用2-ΔΔCt方法[18]计算STK基因的相对表达量。附图1
新窗口打开|下载原图ZIP|生成PPT附图1苗期蘸根法接种黄萎病菌孢子悬浮液
a: 棉苗水培培养。b: 蘸根法接黄萎病菌。
Fig. S1Seedlings were inoculated by V. dahliae via dipping root method in conidial suspension
a: cultivation of cotton seedlings under hydroponic conditions. b: the seedlings were inoculated by V. dahliae.
Table 1
表1
表1试验中所用引物
Table 1
| 引物名称 Primer name | 序列 Primer sequence (5°-3°) | 用途 Purpose |
|---|---|---|
| STK-F1 | CCATATGAAGAATTCAAGCAAATCCTC | 基因扩增Gene amplification |
| STK-R1 | GCTGCAGTCAAGGGGCCGTAGGTTGT | 基因扩增Gene amplification |
| VF | ACTAGTGCTGCTCGAACTTCTATTGGGAAG | 基因扩增Gene amplification |
| VR | GGCGCGCCAGGGGCCGTAGGTTGTGTAACTCT | 基因扩增Gene amplification |
| STK-F2 STK-R2 | ATGCGGCAACGAACAAGAAT TAGATTGGCACTGAGCTGGT | 实时定量qPCR 实时定量qPCR |
| GhUBQ14-F | CAACGCTCCATCTTGTCCTT | 实时定量qPCR |
| GhUBQ14-R | TGATCGTCTTTCCCGTAAGC | 实时定量qPCR |
| BD-F | TTTGTAATACGACTCACTATAGGGCG | 通用载体Vector |
| BD-R | TTTTCGTTTTAAAACCTAAGAGTCAC | 通用载体Vector |
| T7 | TAATACGACTCACTATAGGGCG | 通用载体Vector |
| 3°AD | AGATGGTGCACGATGCACAG | 通用载体Vector |
| TUB2-F | ATCCGTGAAGAGTACCCAGAT | 实时定量qPCR |
| TUB2-R | AAGAACCATGCACTCATCAGC | 实时定量qPCR |
| FT-F | CTTGGCAGGCAAACAGTGTATGCAC | 实时定量qPCR |
| FT-R | GCCACTCTCCCTCTGACAATTGTAGA | 实时定量qPCR |
| SOC1-F | AGCTGCAGAAAACGAGAAGCTCTCTG | 实时定量qPCR |
| SOC1-R | GGGCTACTCTCTTCATCACCTCTTCC | 实时定量qPCR |
新窗口打开|下载CSV
1.3 棉花中病毒诱导的STK基因的沉默
根据GbSTK基因的序列信息, 设计病毒诱导的基因沉默载体(VIGS)引物VF/VR (表1), 并克隆目的片段。将目的基因片段和pTRV2载体分别用Spe I和Asc I进行双酶切, 分别胶回收基因条带和pTRV2载体, 用T4连接酶在16℃过夜连接, 构建成沉默载体pTRV2-GbSTK (TRV::GbSTK)。将连接产物转化大肠杆菌DH5α感受态细胞, 通过PCR检测和阳性克隆测序鉴定得到序列正确的克隆。将构建好的pTRV2-GbSTK质粒和pTRV1分别转化农杆菌GV3101, 保存备用。种植抗病农大棉601, 待棉苗2片子叶平展但第1片真叶未长出时, 分别沉默TRV::GbSTK (试验组)、TRV::00 (空载体对照组)和TRV::CLA1 (环境对照组), 具体操作参照Zhang等[19]方法。待沉默TRV::CLA1的植株新生叶片出现白化, 说明目的基因被有效沉默, 此时, 进一步通过qPCR确定试验组植株的沉默效率。分别选取沉默效果好、长势整齐一致的植株, 按照Zhang等[20]的方法对GbSTK沉默植株和对照植株接种黄萎病菌孢子悬浮液(×107 spores mL-1), 在接菌20 d和25 d后观察植株发病情况并计算病情指数。
1.4 转STK基因拟南芥早花性状的鉴定
分别种植转STK基因拟南芥纯合株系和空载体对照拟南芥, 定期观察拟南芥的长势, 记录拟南芥莲座叶的数目和开花时间。用EASYspin Plus植物RNA提取试剂盒提取转基因株系和空载体对照拟南芥的总RNA, 以TUB2为内参基因(表1), 利用qRT-PCR检测拟南芥中开花相关标志基因FT和SOC1的表达量, 比较转基因植株和对照拟南芥开花途径标志基因的表达量。1.5 STK酵母双杂交载体的构建与转化
将含有正确GbSTK序列信息的质粒和pGBKT7载体分别用Nde I和Pst I进行双酶切, 胶回收GbSTK基因条带和pGBKT7空载体条带, 借助于T4连接酶在16℃过夜连接, 将连接产物转化大肠杆菌DH5α感受态细胞。利用pGBKT7载体的通用引物BD-F/BD-R (表1)检测阳性克隆, 并对PCR阳性克隆提取质粒DNA进一步测序验证。挑选测序正确的克隆, pGBKT7-GbSTK提取质粒DNA, 按照说明书转化Y2HGold 感受态细胞。1.6 诱饵蛋白毒性和自激活检测
参照Matchmaker Gold Yeast Two-Hybrid System操作手册, 以共转pGBKT7-53+pGADT7-T酵母菌作为阳性对照, 共转化pGBKT7-Lam+pGADT7-T酵母菌作为阴性对照。将空载体pGBKT7和pGBKT7-GbSTK分别转入Y2HGold酵母菌株中, 涂于SD/-Trp平板上, 置于30℃培养箱倒置培养3~5 d, 检测诱饵载体pGBKT7-GbSTK有无毒性[21]。挑取平板上生长状态良好的pGBKT7-GbSTK单克隆, 在SD/-Trp液体培养基中培养, 涂布于SD/-Trp、SD/-Trp/X-α-Gal、SD/-Trp/X-α-Gal/AbA平板上, 置于30℃培养箱倒置培养3~5 d, 检测诱饵载体pGBKT7-GbSTK有无自激活活性[21]。
参照Clontech公司酵母双杂交操作指南, 将携带pGBKT7-GbSTK诱饵载体的Y2HGold与黄萎病菌诱导海岛棉酵母双杂交cDNA文库进行杂交, 依次涂布于DDO (SD/-Leu/-Trp)、TDO (SD/-His-/Leu/- Trp or SD/-Ade/-Leu/-Trp)、QDO/X/A (SD/-His-/Ade/- Leu/-Trp/X-α-Gal/AbA)平板上, 置于30℃培养箱倒置培养3~5 d, 观察菌落生长情况和菌落颜色变化。
利用pGADT7通用载体引物T7/3°AD (表1)对所筛选到的单克隆进行PCR鉴定, 对获得的PCR阳性克隆进行测序, 测序结果在Cotton FGD (
2 结果与分析
2.1 不同抗性棉花品种中STK基因的表达
选取不同抗性棉花品种, 根据STK在接菌后12 h出现表达峰值[15], 对根中STK基因的表达检测结果表明, STK基因在不同抗性品种中均有表达, 在感病品种邯208中表达量最低, 不同抗性的棉花品种中STK基因表达量不同程度的明显高于邯208, 其中抗病品种农大棉8号表达量最高, 其次是抗病品种农大601 (ND601), 在农大棉7号(NDM7)、农大棉10号(NDM10)、农大棉13号(NDM13)、国欣棉9号(GXM9)、宁棉9号(NM9)、冀棉169 (JM169)共6个耐病品种中的表达也呈现较高的表达水平(图1)。黄萎病菌诱导下, STK基因的表达水平在抗性好的棉花品种显著高于感病品种, 说明STK基因的转录水平与棉花抗黄萎病反应呈正相关关系。图1
新窗口打开|下载原图ZIP|生成PPT图1棉花不同抗病品种中STK基因的表达
Fig. 1Expression levels of GbSTK gene in different resistant cottons
2.2 沉默棉花内源STK基因显著增强了植株感病性
以抗病农大601为材料, 待两片子叶完全展平且第1片真叶未长出时进行VIGS处理, 以沉默CLA1为可视化对照。VIGS处理7 d后, 注射CLA1的棉苗第1片真叶出现网格状的白化现象(图2-a), 说明基因被VIGS体系有效沉默。此时取沉默棉株及对照棉株的第1片真叶提取RNA, qRT-PCR检测结果表明, 沉默棉株中GbSTK基因的表达量明显低于对照(图2-b), 说明GbSTK在棉株中被沉默。对沉默棉株及对照棉株进行黄萎病菌胁迫处理。在接菌处理25 d的病情指数调查结果表明, 沉默棉株比对照植株表现出明显的叶片黄化、植株萎蔫的典型黄萎病症状(图2-c), 2次独立的基因沉默试验中对照棉株的病情指数分别为25.7和29.3 (平均27.5), 而沉默STK基因的棉株的病情指数分别65.3和61.1 (平均63.2) (图2-d~e)。表明棉花STK正向调控了棉花对黄萎病的抗性。图2
新窗口打开|下载原图ZIP|生成PPT图2在农大601中沉默STK基因对棉花黄萎病抗性的影响
a: 沉默棉花GhCLA1基因; b: TRV::GbSTK植株中STK基因的沉默效率检测; c: 接菌后25 d对照植株与沉默植株的黄萎病抗性表现; d和e: 接菌后25 d对照植株与沉默植株的发病级别调查和病情指数。
Fig. 2Effects of silencing of STK in cotton (ND601) susceptibility to Verticillium dahliae
a: silencing of GhCLA1 gene in cotton; b: qPCR analysis of the STK transcripts in control and silencing seedlings; c: disease symptoms inoculated with V. dahliae on TRV::00 and TRV::GbSTK plants at 25 days; d and e: the rate of diseased plants and disease index were measured at 25 days.
2.3 GbSTK促进转基因拟南芥提早开花
相同生长条件下, 定期观察STK基因拟南芥纯合株系和空载体对照拟南芥的长势, 记录拟南芥莲座叶的数目和开花时间。结果显示, 转基因拟南芥比对照提早开花5~7 d (图3-a~c)。开花时统计转基因株系和野生型拟南芥的莲座叶数目发现, 转基因拟南芥叶片数平均为7.5片, 明显少于野生型的莲座叶片数(11.9片) (图3-d), 表明转GbSTK基因可显著提早拟南芥的开花时间。进一步利用qRT-PCR检测拟南芥开花标志基因FT、SOC1和LFY基因的表达量发现, 转基因不同株系中FT和SOC1基因的表达水平显著高于对照植株, 而LFY基因的表达受影响较小(图3-e~g), 表明STK基因可以影响开花关键基因FT和SOC1的表达水平, 进而在调控植株开花时间中发挥作用。图3
新窗口打开|下载原图ZIP|生成PPT图3过表达GbSTK拟南芥表型鉴定及开花标志基因的表达分析
a~c: 过表达GbSTK基因促进拟南芥提早开花; d: 开花时转基因植株与对照植株莲座叶片数比较; e~g: 转基因植株与对照植株中SOC1、FT和LFY基因的表达。误差线表示标准差, *, ***分别表示在0.05和0.001水平显著差异。WT: 野生型拟南芥; OE: 转GbSTK基因拟南芥; OE1和OE2: 转GbSTK基因拟南芥株系。
Fig. 3Phenotypic evaluation of overexpression of GbSTK in Arabidopsis and expression analysis of the flowering marker genes
a-c: overexpression (OE) of GbSTK in Arabidopsis led to early flowering; d: the transgenic Arabidopsis showed significantly fewer rosette leaves when flowering; e-g: the transgenic Arabidopsis showed significantly higher expression of SOC1 and FT, rather than LFY. Error bars indicate standard error (SE). *, *** indicate significant difference at the 0.05 and 0.001 probability levels, respectively. WT: wild type Arabidopsis; OE: GbSTK transgenic Arabidopsis; OE1 and OE2: different GbSTK transgenic Arabidopsis lines.
2.4 pGBKT7-GbSTK毒性和自激活检测
利用pGBKT7载体的通用引物BD-F/BD-R (表1)检测获得诱饵载体阳性克隆(图4-a~c), 提取阳性克隆质粒DNA测序验证, 测序结果与预期结果大小一致。将空载体对照pGBKT7与诱饵载体pGBKT7-GbSTK分别转化Y2HGold感受态, 涂布于SD/-Trp平板上。在30℃培养箱倒置培养3~5 d后发现, 诱饵载体的平板与空载体的平板菌落生长状态基本一致(图4-d), 表明诱饵载体pGBKT7-GbSTK没有毒性。图4
新窗口打开|下载原图ZIP|生成PPT图4诱饵载体pGBKT7-GbSTK的构建与毒性检测
M: DNA marker DL5000; 1~2: GbSTK-T双酶切; 3~6: pGBKT7双酶切; 7: pGBKT7-GbSTK阳性克隆。d: 诱饵载体pGBKT7- GbSTK毒性检测。
Fig. 4Construction of bait vector of pGBKT7-GbSTK and identification of toxicity
M: DNA marker DL5000; 1-2: double digestion of GbSTK-T vector; 3-6: double digestion of pGBKT7 vector; 7: screening pGBKT7- GbSTK positive clones. d: detection of toxicity for the bait vector of pGBKT7-GbSTK.
自激活试验表明, 阳性对照pGBKT7-53/pGADT7-T在SD/-Leu/-Trp平板上可以长出白色菌落(图5-a), 在SD/-Ade/-His/-Leu/-Trp/X-α-Gal长出蓝色菌落(图5-b), 阴性对照pGBKT7-Lam/ pGADT7-T只能在SD/-Leu/-Trp平板上生长, pGBKT7-GbSTK与阴性对照一致。而且诱饵载体pGBKT7-GbSTK在SD/-Trp/X-α-Gal上正常生长且不显示蓝色, 在SD/-Trp/X-α-Gal/AbA上不能生长(图5-c), 表明pGBKT7-GbSTK没有自激活活性。
图5
新窗口打开|下载原图ZIP|生成PPT图5诱饵载体pGBKT7-GbSTK自激活检测
a: SD/-Leu/-Trp培养基酵母生长情况; b: SD/-Ade/-His/-Leu/-Trp/X-α-Gal培养基酵母生长情况; c: 诱饵载体pGBKT7-GbSTK在培养基中生长情况。
Fig. 5Auto-activation for bait vector of pGBKT7-GbSTK
a: yeast growth on SD/-Leu/-Trp agar plate; b: yeast growth on SD/-Ade/-His/-Leu/-Trp/X-α-Gal agar plate; c: bait vector of pGBKT7- GbSTK growth on agar plate.
2.5 pGBKT7-GbSTK互作蛋白筛选与分析
将携带诱饵载体pGBKT7-GbSTK的酵母菌液和黄萎病菌诱导的海岛棉Pima90-53 cDNA文库杂交, 将菌液依次涂布于DDO、TDO、QDO/X/A平板上, 在30℃培养箱倒置培养3~5 d, 最终挑选生长良好的蓝色菌落(图6: 部分筛选结果), 进行菌落PCR鉴定, 获得105个阳性克隆, 对105个克隆进行测序, 序列信息通过棉花Cotton FGD (图6
新窗口打开|下载原图ZIP|生成PPT图6筛选阳性克隆
Fig. 6Screening of positive clones
Table 2
表2
表2与GbSTK互作的候选蛋白
Table 2
| 编号 No. | 蛋白描述 Protein description | 功能报道 Function reported | 功能分类 Functional classification |
|---|---|---|---|
| 1 | Cytochrome c oxidase subunit 6b-1 | Regulate ROS production[22] | ROS production |
| 2 | Malate dehydrogenase | Related to production of ROS and PCD[23] | |
| 3 | GTPase-activating protein GYP7 | Defense response[24] | Involved in defense and abiotic/biotic stresses |
| 4 | Cytochrome P450 71D8 | Defense response[25] | |
| 5 | Short-chain dehydrogenase reductase 3b | Involved in plant defense responses[26] | |
| 6 | Cysteine proteinase RD21a | Involved in plant defense[27] | |
| 7 | Vacuolar protein sorting-associated protein 55 | Involved in plant defense[28] | |
| 8 | Pathogenesis-related protein STH-2 | Defense response[29] | |
| 9 | (-)-alpha-terpineol synthase | Involved in defense and abiotic stresses[30] | |
| 10 | Glyceraldehyde-3-phosphate dehydrogenase 2 | Negatively regulate autophagy and immunity[31] | |
| 11 | Phosphoglycerate Ubiquitin-conjugating enzyme E2 | Involved in plant immunity[32] | |
| 12 | Dirigent protein 16 | Responsive to biotic stress[33] | |
| 13 | Major allergen Pruar 1 | Novel Pathogenesis-related protein[34] | |
| 14 | S-Adenosylmethionine synthase | Responsive to abiotic stress[35] | |
| 15 | S-adenosylmethionine synthase 2 | Responsive to abiotic stress[36] | |
| 16 | Probable inorganic phosphate transporter 1-5 | Involved in phosphate homeostasis[37] | |
| 17 | Glycerophosphodiester phosphodiesterase GDPD1 | Response to Pi deficiency[38] | |
| 18 | Delta-cadinene synthase isozyme C2 | Gossypol biosynthetic[39] | Secondary metabolism and synthesis |
| 19 | UDP-galactose/UDP-glucose transporter 3 | Synthesis of cell walls[40] | |
| 20 | Aldehyde dehydrogenase family 2 member C4 | Regulator of the growth-defense trade-off[41] | Plant development and stress response |
| 21 | Probable 2-oxoglutarate-dependent dioxygenase | Related to vascular disease/involved in gibberellin synthesis[42] | |
| 22 | Patellin-5 | Function in plant development and stress response[43] | |
| 23 | Triphosphate tunel metalloenzyme 3 | Catalyze diverse enzymatic reactions[44] | |
| 24 | Chloroplastic 2,3-bisphosphoglycerate-independent mutase | Involved in photosynthesis and chloroplast development[45] | Plant growth and development |
| 25 | Synaptotagmin-2 | Participates in pollen germination and tube growth[46] | |
| 26 | Mediator of RNA polymerase II transcription subunit | — | |
| 27 | Alpha-taxilin | — | |
| 28 | Aconitate hydratase, cytoplasmic | — | |
| 29 | Putative zinc transporter At3g08650 | — | |
| 30 | 40S ribosomal protein S5 | — |
新窗口打开|下载CSV
3 讨论
自然条件下, 植物生长发育面临生物和非生物胁迫等复杂的环境, 植株为了增强生存能力, 在长期的植物进化过程中形成了一套成熟的适应机制, 即通过能量和物质的重新分配来有效调控生长与发育的平衡。传统观点认为, 植株抗病性与品种早熟性存在此消彼长的关系, 认为抗病植株易贪青晚熟, 早熟性好的品种抗性比较差, 犹如鱼和熊掌不可兼得[47]。随着人们对基因调控网络的不断建立和深入, 一些突破性研究进展正在冲击人们的传统认识, 正如普遍认为植物抗病与产量之间呈现难以打破的负相关, 最新发表在Science的研究报道表明[48], 水稻IPA1基因既能提高水稻产量又能增强对稻瘟病的抗性, 打破了不可能同时实现增产和抗病的传统观点, 这也为育种家突破传统认识, 将之前多年来育种上的不可能逐渐变为可能提供了启发。本研究发现, 棉花丝氨酸苏氨酸蛋白激酶(GbSTK)具有调节黄萎病抗性和植株早花的双重功能, 为从理论上打破抗病性与品种早熟性负相关提供了有潜力的基因资源。本课题组前期基于SSH文库筛选到GbSTK基因, 该基因可以显著提高拟南芥的黄萎病抗性[15]。本研究在此基础上进一步研究发现, 抗性较好的多个棉花品种中STK基因表达水平均不同程度高于感病对照, VIGS试验进一步证明, 沉默棉花内源STK基因显著降低了植株的抗病性, 表明GbSTK正调控棉花黄萎病抗性。酵母双杂交互作蛋白筛选试验初步筛选到30个与GbSTK存在互作关系的蛋白, 大多数蛋白的功能尚未报道。其中, 我们筛选到的S-腺苷甲硫氨酸合成酶(S-adenosylmethionine synthase, SAMS)目前已有研究。SAMS是植物代谢中的关键酶, 催化甲硫氨酸和ATP合成S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)[49]。SAM不仅为DNA、RNA、蛋白质提供了甲基, 而且还是多胺、乙烯、生物素等物质合成的前体物质[50,51]。表明SAMS基因参与植物响应生物与非生物胁迫。在拟南芥中, FER通过与SAM1和SAM2酶相互作用, 抑制了SAM的合成, 降低了乙烯水平, 从而控制了植物的发育[52]。大豆在水分和干旱胁迫下, SAMS参与应激反应调控, 3种S-腺苷甲硫氨酸合成酶蛋白在水分和干旱胁迫下表达分别下降、增加[53]。张悦等[54]研究表明, 刚毛柽柳S-腺苷甲硫氨酸合成酶基因ThSAMS参与了对盐和干旱的胁迫应答。董先娟等[55]发现, 盐、干旱、低温以及重金属胁迫均能够提高AsSAMS1的表达和SAM的积累; 茉莉酸甲酯、水杨酸、脱落酸、赤霉素信号均可诱导白木香愈伤组织中AsSAMS1基因的表达。本课题组也发现, 陆地棉GhSAMS基因在棉花抗黄萎病反应中起着重要作用[35]。本研究基于酵母双杂交筛选的与GbSTK互作的SAMS蛋白和GhSAMS蛋白的同源性为99.28%推测(附图2), GbSTK蛋白可能通过与SAMS蛋白互作, 从而起到抗黄萎病作用, 也为深入研究GbSTK蛋白的抗病机制奠定了基础。
附图2
新窗口打开|下载原图ZIP|生成PPT附图2SAMS蛋白和陆地棉GhSAMS蛋白同源性分析
Fig. S2Homology analysis of SAMS protein and GhSAMS protein
FLOWERING LOCUS T(FT)基因在植物中广泛存在, 作用于多种调控途径的下游, 包括CONSTANS (CO)光周期诱导途径, 是促进开花的所必需的信号整合因子[56]。CO蛋白在长日光条件下激活FT基因表达, FT蛋白被运送到筛管中, 最终通过韧皮部运输到茎尖分生组织和FD蛋白结合, 共同作用调控下游花器官相关基因AP1、SOC1等基因的表达, 从而促进拟南芥开花[57]。本研究发现, 转GbSTK基因的拟南芥比对照提早开花5~7 d, qRT-PCR分析拟南芥开花关键基因的表达水平发现, GbSTK基因可以显著提高拟南芥FT和SOC1基因的转录水平, 促使植株提早开花, 这也首次揭示了一个具有抗病功能的棉花基因具有提早植株开花的功能。
本研究初步证明了棉花GbSTK基因具有同时提高抗性和提早开花的功能, 并为进一步研究该基因的分子机制提供了初步线索, 深入研究GbSTK基因的功能和分子机制, 将为抗病早熟育种提供重要理论基础和实际应用新途径。
4 结论
GbSTK基因正向调控棉花黄萎病抗性, 沉默棉花内源STK基因可显著降低植株抗病性; 超表达GbSTK可以增强开花关键基因FT和SOC1的表达, 进而调控植株提早开花。酵母双杂交初步筛选出30个与GbSTK互作的候选蛋白, 主要涉及参与ROS合成、生物非生物胁迫、次生代谢、生长发育等几类功能蛋白。参考文献 原文顺序
文献年度倒序
文中引用次数倒序
被引期刊影响因子
DOI:10.1073/pnas.0507766103URLPMID:16477027 [本文引用: 1]
Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 A), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 A) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors.
[本文引用: 1]
[本文引用: 1]
DOI:10.1006/bbrc.2001.6269URLPMID:11785954 [本文引用: 1]
Previously, we reported the identification of a gibberellin (GA)-binding protein in rice using ligand binding assay that was homologous to RuBisCO activase (Komatsu et al., FEBS Lett. 384, 167-171, 1996). Here, we provide an evidence for the involvement of protein kinases components downstream to the GA-binding phosphoprotein, RuBisCO activase in rice. Ca(2+)-dependent protein kinase activity was studied in subcellular fractions of leaf sheath from transgenic rice containing sense and antisense constructs of RuBisCO activase. In-gel kinase assay using histone III-S as a substrate showed constitutive induction of a 46- and 48-kDa Ca(2+)-dependent protein kinase activity in the sense transgenic plants. Kinase activities of these proteins were significantly reduced in the presence of uniconazole, a potent GA biosynthesis inhibitor, but one of them was strongly promoted by GA(3) treatment in transgenic plants carrying a smaller subunit of RuBisCO activase (OsrcaA1) compared to the larger subunit OsrcaA2. Also, in vitro phosphorylation studies using two-dimensional polyacrylamide gel showed changes in the degree of phosphorylation of several proteins in OsrcaA1- and OsrcaA2-sense transgenic rice. These studies suggest the presence of two independent cytosolic Ca(2+)-dependent protein kinase signaling components downstream to the GA-binding protein in rice suggesting their role in GA signaling.
DOI:10.1007/s10482-013-9993-2URLPMID:23918348 [本文引用: 1]
The regulation of signal transduction by phosphorylation and ubiquitination is essential to ensure the correct behavior of eukaryotic cells. We searched for protein families involved in such signaling in several eukaryotic species and in a limited set of prokaryotes, where two members of the Planctomycetes phylum were included as they exhibit eukaryote-like features (Gemmata obscuriglobus and Pirellula staleyi). We identified sequences homologous to eukaryotic serine/threonine kinases (STKs) and E2-ubiquitin conjugating enzymes in the two Planctomycetes species. To extend these analyses to the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum, we performed comparative analyses using domains from kinases, phosphatases and GTPases that serve as signaling signatures, and we analyzed their distributions. We found substantial differences in kinome densities with regards to other prokaryote clades and among the groups in the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum. In addition, we identified the presence of classic eukaryotic E2-conjugating ubiquitin proteins in prokaryotes, these having previously believed to exist only in eukaryotes. Our phylogenetic analyses of the STKs signature domains and E2-enzymes suggest the existence of horizontal gene transfer.
DOI:10.1016/s0092-8674(02)00743-2URLPMID:12062102 [本文引用: 1]
The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.
URLPMID:7902614 [本文引用: 1]
DOI:10.1016/j.plantsci.2019.110318URLPMID:31779898 [本文引用: 1]
Plant receptor-like kinase (RLKs) are serine/threonine protein kinases that play fundamental roles in development, innate immunity, and abiotic stress response. Here, we identified an S-domain receptor-like kinase OsESG1 from rice (Oryza sativa), and identified its involvement in early crown root (CR) development and drought response. The OsESG1 kinase domain possessed auto-phosphorylation activity and was able to phosphorylate MBP and His proteins. OsESG1 was expressed ubiquitously in all tissues that were examined, with relatively higher expression in the embryo. And it could be induced to express by treating with PEG, NaCl and ABA. Transgenic plants carrying anti-sense (AS) OsESG1 were generated by knockdown of OsESG1 expression. At the early seedling stage, AS lines had fewer CRs and shorter shoot compared with wild type (WT) plants. IAA flux and the genes' expressions of the auxin responsive and efflux carrier were infected in the AS lines. These results indicated that auxin signaling and polar auxin transport (PAT) were disrupted. The AS lines were more sensitive to osmotic stress compared to WT, and showed excessive accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), lower activities of antioxidant enzymes, and impaired expressions of stress-related genes under PEG treatment. Results above suggested that OsESG1 may regulate CR initiation and development by controlling auxin response and distribution, and participate in stress response by regulating the activities of antioxidants and expressions of stress-regulated genes.
DOI:10.1126/science.270.5243.1804URLPMID:8525370 [本文引用: 1]
The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.
[本文引用: 1]
[本文引用: 1]
URLPMID:31527679 [本文引用: 1]
URLPMID:21508323 [本文引用: 1]
[本文引用: 1]
[本文引用: 1]
DOI:10.1007/BF00279745URLPMID:7845351 [本文引用: 1]
A cDNA clone (cNPK15) was isolated from tobacco cells in suspension culture, which encodes a predicted protein kinase of 422 amino acids. The predicted NPK15 protein consists of a hydrophobic region near the amino-terminus, a linker domain and the catalytic domain of a protein-serine/threonine kinase in the carboxyl-half. NPK15 was not found to be closely related to any reported protein, but its putative catalytic domain shares some structural similarity with those of receptor-like protein kinases of plants, such as ZmPK1 from Zea mays and TMK1 from Arabidopsis, even though no receptor-like domain is found in NPK15. Recombinant NPK15 expressed in Escherichia coli as a fusion protein was found capable of autophosphorylation and of phosphorylation of the histone H1 protein on both serine and threonine residues. Upon overexpression of cNPK15 under control of the promoter of cauliflower mosaic virus 35S RNA in tobacco cells, into which it had been introduced by Agrobacterium-mediated transformation, the NPK15 gene acted as a
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[本文引用: 1]
URLPMID:23912851 [本文引用: 4]
[本文引用: 1]
[本文引用: 1]
DOI:10.3389/fpls.2016.01830URLPMID:28018374 [本文引用: 1]
Cotton is one of the most economically important crops, but most cultivated varieties lack adequate innate immunity or resistance to Verticillium wilt. This results in serious losses to both yield and fiber quality. To identify the genetic resources for innate immunity and understand the pathways for pathogen defenses in this crop, here we focus on orthologs of the central Arabidopsis thaliana defense regulator Enhanced Disease Susceptibility 1 (EDS1). The full-length cDNA of GbEDS1 was obtained by screening the full-length cDNA library of Gossypium barbadense combining with RACE strategy. Its open reading frame is 1848 bp long, encoding 615 amino acid residues. Sequence analysis showed that GbEDS1 contains a conserved N-terminal lipase domain and an EDS1-specific KNEDT motif. Expression profiling indicated that the gene is induced by Verticillium dahliae as well as salicylic acid (SA) treatment. Subcellular localization assays revealed that GbEDS1 is located in the cell cytoplasm and nucleus. Overexpression of GbEDS1 in Arabidopsis dramatically up-regulated SA and H2O2 production, resulting in enhanced disease resistance to V. dahliae. Silencing of GbEDS1 in G. barbadense significantly decreased SA and H2O2 accumulation, leading to the cotton more susceptibility. Moreover, combining the gene expression results from transgenic Arabidopsis and silenced-GbEDS1 cotton, it indicated that GbEDS1 could activate GbNDR1 and GbBAK1 expression. These findings not only broaden our knowledge about the biological role of GbEDS1, but also provide new insights into the defense mechanisms of GbEDS1 against V. dahliae in cotton.
DOI:10.1006/meth.2001.1262URLPMID:11846609 [本文引用: 1]
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
DOI:10.1111/mpp.12755URLPMID:30267563 [本文引用: 1]
Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The lignification of cell wall appositions is a conserved basal defence mechanism in the plant innate immune response. However, the function of laccase in defence-induced lignification has not been described. Screening of an SSH library of a resistant cotton cultivar, Jimian20, inoculated with V. dahliae revealed a laccase gene that was strongly induced by the pathogen. This gene was phylogenetically related to AtLAC15 and contained domains conserved by laccases; therefore, we named it GhLAC15. Quantitative reverse transcription-polymerase chain reaction indicated that GhLAC15 maintained higher expression levels in tolerant than in susceptible cultivars. Overexpression of GhLAC15 enhanced cell wall lignification, resulting in increased total lignin, G monolignol and G/S ratio, which significantly improved the Verticillium wilt resistance of transgenic Arabidopsis. In addition, the levels of arabinose and xylose were higher in transgenic plants than in wild-type plants, which resulted in transgenic Arabidopsis plants being less easily hydrolysed. Furthermore, suppression of the transcriptional level of GhLAC15 resulted in an increase in susceptibility in cotton. The content of monolignol and the G/S ratio were lower in silenced cotton plants, which led to resistant cotton cv. Jimian20 becoming susceptible. These results demonstrate that GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum. This study broadens our knowledge of defence-induced lignification and cell wall modifications as defence mechanisms against V. dahliae.
URLPMID:21739145 [本文引用: 1]
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[本文引用: 2]
DOI:10.1007/s00109-020-01905-yURLPMID:32313986 [本文引用: 1]
Psychosocial stress is known to cause an increased incidence of coronary heart disease. In addition, multiple other diseases like cancer and diabetes mellitus have been related to stress and are mainly based on excessive formation of reactive oxygen species (ROS) in mitochondria. The molecular interactions between stress and ROS, however, are still unknown. Here we describe the missing molecular link between stress and an increased cellular ROS, based on the regulation of cytochrome c oxidase (COX). In normal healthy cells, the
DOI:10.1038/s41422-018-0024-8URLPMID:29540758 [本文引用: 1]
Programmed cell death (PCD) is a fundamental biological process. Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase, leads to the accumulation of reactive oxygen species (ROS) and PCD, which can be suppressed by mitochondrial complex I mutations, indicating a signal from chloroplasts to mitochondria. However, this signal remains to be elucidated. In this study, through cloning and analyzing a series of mod1 suppressors, we reveal a comprehensive organelle communication pathway that regulates the generation of mitochondrial ROS and triggers PCD. We show that mutations in PLASTIDIAL NAD-DEPENDENT MALATE DEHYDROGENASE (plNAD-MDH), chloroplastic DICARBOXYLATE TRANSPORTER 1 (DiT1) and MITOCHONDRIAL MALATE DEHYDROGENASE 1 (mMDH1) can each rescue the ROS accumulation and PCD phenotypes in mod1, demonstrating a direct communication from chloroplasts to mitochondria via the malate shuttle. Further studies demonstrate that these elements play critical roles in the redox homeostasis and plant growth under different photoperiod conditions. Moreover, we reveal that the ROS level and PCD are significantly increased in malate-treated HeLa cells, which can be dramatically attenuated by knockdown of the human gene MDH2, an ortholog of Arabidopsis mMDH1. These results uncover a conserved malate-induced PCD pathway in plant and animal systems, revolutionizing our understanding of the communication between organelles.
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DOI:10.1016/j.plaphy.2011.10.013URLPMID:22153241 [本文引用: 1]
ABSCISIC ACID DEFICIENT2 (ABA2) encodes a short-chain dehydrogenase/reductase1 (SDR1) that catalyzes the multi-step conversion of xanthoxin to abscisic aldehyde during abscisic acid (ABA) biosynthesis in Arabidopsis thaliana. In this study, AtSDR2 and AtSDR3, the two closest homologs to AtABA2, were investigated for their potential role in ABA biosynthesis. AtSDR2 showed undetectable transcription in plants grown under normal conditions or under stress. AtSDR3 and AtABA2 have different spatial and temporal expression patterns. Complementation testing demonstrated that the pABA2::SDR3 transgene failed to complement the aba2 mutant phenotype, and that transgenic plants showed the same levels of ABA as the aba2 mutants. These data suggest that AtSDR3 confers no functional redundancy to AtABA2 in ABA biosynthesis. Interestingly, microarray data derived from Genevestigator suggested that AtSDR3 might have a function that is related to plant defense. Pseudomonas syringae pv. tomato (Pst) DC3000 infection and systemic acquired resistance (SAR) activator application further demonstrated that AtSDR3 plays an important role in plant defense responses at least partially through the regulation of AtPR-1 gene expression.
DOI:10.1007/s00299-019-02502-1URLPMID:31993730 [本文引用: 1]
KEY MESSAGE: Transgenic rice overexpressing PLCP attenuated the virulence of Xanthomonas oryzae pv. oryzae through extensive activation of transduction signal and transcription activities that orchestrate downstream responses including the biosynthesis of secondary metabolites and up-regulation of several pathogenesis-related proteins. High-throughput transcriptome investigations of plant immunity highlight the complexity of gene networks leading to incompatible interaction with the pathogen. Accumulating findings implicate papain-like cysteine proteases (PLCPs) as a central hub in plant defense. While diverse roles of PLCPs in different pathosystems have become more evident, information on gene networks and signaling pathways necessary to orchestrate downstream responses are lacking. To understand the biological significance of cysteine protease against Xanthomonas oryzae pv. oryzae, PLCP overexpression and knockout rice lines were generated. The pathogenicity test revealed the attenuation of Xanthomonas oryzae pv. oryzae race K3a virulence in transgenic lines which is ascribed to high hydrogen peroxide and free salicylic acid accumulation. Next-generation sequencing of RNA from transgenic and wild-type plants identified 1597 combined differentially expressed genes, 1269 of which were exclusively regulated in the transgenic libraries. It was found that PLCP aids rice to circumvent infection through the extensive activation of transduction signal and transcription factors that orchestrate downstream responses, including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites.
DOI:10.1007/s12275-015-5067-7URLPMID:25845538 [本文引用: 1]
Fusarium graminearum, a member of the F. graminearum species complex, is a filamentous ascomycetous group that causes serious diseases in cereal crops. A screen of insertional mutants of F. graminearum, generated using a restriction enzyme-mediated integration method, identified a mutant designated R7048 showing pleiotropic phenotypes in several mycological traits. The vector insertion site in the R7048 genome was identified as the KpnI site within an ORF annotated as FGSG_06346 (designated FgVPS74), which showed similarity to vacuolar protein sorting-associated protein 74 in the baker yeast. Both targeted gene deletion and complementation analyses confirmed that FgVPS74 was involved in hyphal growth, conidiation, sexual development, mycotoxin production, and virulence towards host plants in F. graminearum. Electron microscopy analysis revealed no significant changes in morphology of the vacuole or other organelles, but a greater number of mitochondria were produced in the DeltaFgVPS74 strain compared to the wild-type progenitor. Expression of a GFP-tagged FgVPS74 construct under its native promoter in the DeltaFgVPS74 strain exhibited localization of GFP signal to putative vesicle structures, but not to the vacuolar membrane. Taken together, these findings demonstrated that a functional vacuolar protein-sorting pathway mediated by FgVPS74 is crucial for fungal growth and development in F. graminearum.
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DOI:10.1186/s12870-019-1720-3URLPMID:30922222 [本文引用: 1]
BACKGROUND: It is well known that aromatic essential oils extracted from the heartwood of Santalum album L. have wide economic value. However, little is known about the role of terpenoids in response to various adverse environmental stresses as other plants do in the form of signals during plant-environment interactions. RESULTS: In this study, trace amounts of volatiles consisting of alpha-santalene, epi-beta-santalene, beta-santalene, alpha-santalol, beta-santalol, (E)-alpha-bergamotene, (E)-beta-farnesene and beta-bisabolene were found in the leaves of mature S. album trees. We identified more than 40 candidate terpene synthase (TPS) unigenes by mining publicly-available RNA-seq data and characterized the enzymes encoded by three cDNAs: one mono-TPS catalyzes the formation of mostly alpha-terpineol, and two multifunctional sesqui-TPSs, one of which produces (E)-alpha-bergamotene and sesquisabinene as major products and another which catalyzes the formation of (E)-beta-farnesene, (E)-nerolidol and (E,E)-farnesol as main products. Metabolite signatures and gene expression studies confirmed that santalol content is closely related with santalene synthase (SaSSY) transcripts in heartwood, which is key enzyme responsible for santalol biosynthesis. However, the expression of three new SaTPS genes differed significantly from SaSSY in the essential oil-producing heartwood. Increased activities of antioxidant enzymes, superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, were detected in different tissues of S. album plants after applying 1 mM methyl jasmonate (MeJA) and 1 mM salicylic acid (SA), or exposure to 4 degrees C, 38 degrees C and high light intensity. MeJA and SA dramatically induced the expression of SaTPS1 and SaTPS2 in leaves. SaTPS1 to 3 transcripts were differentially activated among different tissues under adverse temperature and light stresses. In contrast, almost all SaSSY transcripts decreased in response to these environmental stresses, unlike SaTPS1 to 3. CONCLUSIONS: Multifunctional enzymes were biochemically characterized, including one chloroplastic mono-TPS and two cytosolic sesqui-TPSs in sandalwood. Our results suggest the ecological importance of these three new SaTPS genes in defensive response to biotic attack and abiotic stresses in S. album.
DOI:10.1105/tpc.114.134692URLPMID:25829441 [本文引用: 1]
Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity.
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DOI:10.1016/j.jaci.2011.04.020URLPMID:21571358 [本文引用: 1]
BACKGROUND: Allergen-specific immunotherapy for food allergies, including peach allergy, has not been established. Use of allergens with reduced allergenic potential and preserved immunogenicity could improve the safety and efficacy of allergen-specific immunotherapy. OBJECTIVE: We sought to create a hypoallergenic derivative of the major peach allergen Pru p 3 and to characterize its biochemical and immunologic properties. METHODS: A Pru p 3 folding variant generated by means of reduction and alkylation was investigated for structural integrity and stability to gastrointestinal enzymes. IgE reactivity and allergenic potency were determined by means of immunoblotting, ELISA, and in vitro mediator release assay with sera from patients with peach allergy. T-cell immunogenicity was investigated by using human allergen-specific T cells and CBA/J mice immunized with either native Pru p 3 (nPru p 3) or reduced and alkylated (R/A) Pru p 3. Pru p 3 processing by endolysosomal fractions of dendritic cells and antigenicity was examined in mice. RESULTS: Unfolding of Pru p 3 reduced its high resistance to gastrointestinal proteolysis and almost completely abrogated its IgE reactivity and allergenic potency. However, R/A Pru p 3 was capable of stimulating human and murine T cells. Endolysosomal degradation of R/A Pru p 3 was accelerated in comparison with nPru p 3, but similar peptides were generated. IgG and IgE antibodies raised against nPru p 3 showed almost no cross-reactivity with R/A Pru p 3. Moreover, the antigenicity of R/A Pru p 3 was strongly reduced. CONCLUSION: Unfolded Pru p 3 showed reduced allergenicity and antigenicity and preserved T-cell immunogenicity. The hypoallergenic variant of Pru p 3 could be a promising vaccine candidate for specific immunotherapy of peach allergy.
DOI:10.1007/s00425-015-2463-5URLPMID:26757733 [本文引用: 2]
MAIN CONCLUSION: Cotton S-adenosylmethionine decarboxylase-, rather than spermine synthase-, mediated spermine biosynthesis is required for salicylic acid- and leucine-correlated signaling in the defense response to Verticillium dahliae. Spermine (Spm) signaling is correlated with plant resistance to the fungal pathogen Verticillium dahliae. We identified genes for key rate-limiting enzymes in the biosynthesis of Spm, namely S-adenosylmethionine decarboxylase (GhSAMDC) and Spm synthase (GhSPMS). These were found by screening suppression subtractive hybridization and cDNA libraries of cotton (Gossypium) species tolerant to Verticillium wilt. Both were induced early and strongly by inoculation with V. dahliae and application of plant hormones. Silencing of GhSPMS or GhSAMDC in cotton leaves led to a significant accumulation of upstream substrates and, ultimately, enhanced plant susceptibility to Verticillium infection. Exogenous supplementation of Spm to the silenced cotton plants improved resistance. When compared with the wild type (WT), constitutive expression of GhSAMDC in Arabidopsis thaliana was associated with greater Verticillium wilt resistance and higher accumulations of Spm, salicylic acid, and leucine during the infection period. By contrast, transgenic Arabidopsis plants that over-expressed GhSPMS were unexpectedly more susceptible than the WT to V. dahliae and they also had impaired levels of putrescine (Put) and salicylic acid (SA). The susceptibility exhibited in GhSPMS-overexpressing Arabidopsis plants was partially reversed by the exogenous supply of Put or SA. In addition, the responsiveness of those two transgenic Arabidopsis lines to V. dahliae was associated with an alteration in transcripts of genes involved in plant resistance to epidermal penetrations and amino acid signaling. Together, these results suggest that GhSAMDC-, rather than GhSPMS-, mediated spermine biosynthesis contributes to plant resistance against V. dahliae through SA- and leucine-correlated signaling.
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DOI:10.1007/s004250050055URLPMID:10787059 [本文引用: 1]
The cotton (+)-delta-cadinene synthase, a sesquiterpene cyclase, is encoded by a complex gene family which, based on homology, can be divided into two subfamilies: cad1-A and cad1-C. Southern blots revealed several members of the cad1-C subfamily, and a single member of the cad1-A subfamily, in the diploid Gossypium arboreum genome. One of the cad1-C genes, cad1-C3, was isolated from this species. According to reverse transcriptase-polymerase chain reaction, transcripts of both cad1-C and cad1-A genes appeared in roots from the second day post germination and in 1-d-old cotyledons, whereas the transcription levels were too low to be detected in the hypocotyls. Initially, sesquiterpene cyclase activities were found to be high in the seedlings, then dropped in aerial organs but increased in roots during development. Sesquiterpene aldehyde contents followed the same pattern. In fully developed plants, the transcripts of cad1-C were detected in stems, leaves and pericarps, as well as in the sepals and petals 3 d before anthesis, but not at the day of anthesis. In contrast, cad1-A transcripts were not detected in any of these aerial organs. The sesquiterpene aldehyde contents increased in petals but decreased in sepals after anthesis. Treatment of G. arboreum stems with a Verticillium dahliae elicitor-preparation activated cad1-A transcription, but a significant level of cad1-C transcripts was detected both before and after elicitation. In G. hirsutum cv. GL-5, a glandless cultivar, the cad1-C gene was activated by the same fungal elicitor, followed by the synthesis of the sesquiterpene cyclase, and accumulation of sesquiterpene aldehydes. The cad1 gene expression during development and in response to elicitation, as well as the spatial and temporal pattern of sesquiterpene biosynthesis, constitute a chemical defense machinery in cotton plants.
DOI:10.4161/psb.6.8.16379URLPMID:21822061 [本文引用: 1]
Cell wall-related nucleotide sugar transporters (NSTs) theoretically supply the cytosolic nucleotide sugars for glycosyltransferases (GTs) to carry out ploysaccharide synthesis and modification in the Golgi apparatus. However, the regulation of cell wall synthesis by NSTs remains undescribed. Recently, we have reported the functional characterization of Oryza sativa nucleotide sugar transport (Osnst1) mutant and its corresponding gene. OsNST1/BC14 is localized in the Golgi apparatus and transports UDP-glucose. This mutant provides us with a unique opportunity for evaluation of its abroad impacts on cell wall structure and components. We previously examined cell wall composition of bc14 and wild type plants. Here, the spatial distribution of these cell wall alterations was analyzed by immunolabeling approach. Analysis of the sugar yield in different cell wall fractions indicated that this mutation improves the extractability of cell wall components. Field emission scanning electron microscopy further showed that the orientation of microfibrils in bc14 is irregular when compared to that in wild type. Therefore, this UDP-glucose transporter, making substrates available for polysaccharide biosynthesis, plays a critical role in maintaining cell wall integrity.
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DOI:10.1016/j.molp.2015.09.003URLPMID:26384245 [本文引用: 1]
Arabidopsis synaptotagmin 2 (SYT2) has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen of Arabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmMan1-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.
DOI:10.1016/j.molp.2018.10.005URLPMID:30853061 [本文引用: 1]
To optimize fitness, plants must efficiently allocate their resources between growth and defense. Although phytohormone crosstalk has emerged as a major player in balancing growth and defense, the genetic basis by which plants manage this balance remains elusive. We previously identified a quantitative disease-resistance locus, qRfg2, in maize (Zea mays) that protects against the fungal disease Gibberella stalk rot. Here, through map-based cloning, we demonstrate that the causal gene at qRfg2 is ZmAuxRP1, which encodes a plastid stroma-localized auxin-regulated protein. ZmAuxRP1 responded quickly to pathogen challenge with a rapid yet transient reduction in expression that led to arrested root growth but enhanced resistance to Gibberella stalk rot and Fusarium ear rot. ZmAuxRP1 was shown to promote the biosynthesis of indole-3-acetic acid (IAA), while suppressing the formation of benzoxazinoid defense compounds. ZmAuxRP1 presumably acts as a resource regulator modulating indole-3-glycerol phosphate and/or indole flux at the branch point between the IAA and benzoxazinoid biosynthetic pathways. The concerted interplay between IAA and benzoxazinoids can regulate the growth-defense balance in a timely and efficient manner to optimize plant fitness.
DOI:10.1126/science.aat7675URLPMID:30190406 [本文引用: 1]
Plant immunity often penalizes growth and yield. The transcription factor Ideal Plant Architecture 1 (IPA1) reduces unproductive tillers and increases grains per panicle, which results in improved rice yield. Here we report that higher IPA1 levels enhance immunity. Mechanistically, phosphorylation of IPA1 at amino acid Ser(163) within its DNA binding domain occurs in response to infection by the fungus Magnaporthe oryzae and alters the DNA binding specificity of IPA1. Phosphorylated IPA1 binds to the promoter of the pathogen defense gene WRKY45 and activates its expression, leading to enhanced disease resistance. IPA1 returns to a nonphosphorylated state within 48 hours after infection, resuming support of the growth needed for high yield. Thus, IPA1 promotes both yield and disease resistance by sustaining a balance between growth and immunity.
DOI:10.1016/j.plaphy.2019.06.006URLPMID:31238253 [本文引用: 1]
S-adenosylmethionine synthetase (SAMS) catalyzes methionine and ATP to generate S-adenosyl-L-methionine (SAM). In plants, accumulating SAMS genes have been characterized and the majority of them are reported to participate in development and stress response. In this study, two putative SAMS genes (CsSAMS1 and CsSAMS2) were identified in cucumber (Cucumis Sativus L.). They displayed 95% similarity and had a high identity with their homologous of Arabidopsis thaliana and Nicotiana tabacum. The qRT-PCR test showed that CsSAMS1 was predominantly expressed in stem, male flower, and young fruit, whereas CsSAMS2 was preferentially accumulated in stem and female flower. And they displayed differential expression profiles under stimuli, including NaCl, ABA, SA, MeJA, drought and low temperature. To elucidate the function of cucumber SAMS, the full-length CDS of CsSAMS1 was cloned, and prokaryotic expression system and transgenic materials were constructed. Expressing CsSAMS1 in Escherichia coli BL21 (DE3) improved the growth of the engineered strain under salt stress. Overexpression of CsSAMS1 significantly increased MDA content, H2O2 content, and POD activity in transgenic lines under non-stress condition. Under salt stress, however, the MDA content of transgenic lines was lower than that of the wild type, the H2O2 content remained high, the polyamine and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way. Taken together, our results suggested that both CsSAMS1 and CsSAMS2 were involved in plant development and stress response, and a proper increase of expression level of CsSAMS1 in plants is benificial to improving salt tolerance.
DOI:10.1007/s11103-009-9490-1URLPMID:19396585 [本文引用: 1]
S'adenosyl-L: -methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene, polyamines, biotin, and nicotianamine in plants. Only limited information is available regarding its synthesis (SAM cycle) and its concentrations in plant tissues. The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight. Troughs of SAM levels were measured in the evening and night, which corresponds to the time when the major floral scent compound, methyl benzoate, is synthesized by a SAM dependent methyltransferase (NsBSMT) and when this enzyme possesses its highest activity. The SAM synthetase (NsSAMS1) and methionine synthase (NsMS1) are enzymes, among others, which are involved in the synthesis and regeneration of SAM. Respective genes were isolated from a N. suaveolens petal cDNA library. Transcript accumulation patterns of both SAM regenerating enzymes matched perfectly those of the bifunctional NsBSMT; maximum mRNA accumulations of NsMS1 and NsSAMS1 were attained in the evening. Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers. It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).
DOI:10.1111/j.1365-313X.2006.02942.xURLPMID:17144895 [本文引用: 1]
The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.
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