,2,*Suppression mechanism of volatile sprout-inhibitors on potato tuber sprouting
ZOU Xue1,2, DING Fan1, YU Jin-Long1, PENG Jie2, DENG Meng-Sheng2, WANG Yu2, LIU Li-Fang1, YU-HAN Kai-Zong1, CHEN Nian-Wei1, WANG Xi-Yao,2,*通讯作者:
第一联系人:
收稿日期:2018-04-29接受日期:2018-08-20网络出版日期:2018-09-26
| 基金资助: |
Received:2018-04-29Accepted:2018-08-20Online:2018-09-26
| Fund supported: |
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邹雪, 丁凡, 余金龙, 彭洁, 邓孟胜, 王宇, 刘丽芳, 余韩开宗, 陈年伟, 王西瑶. 挥发性抑芽物质对马铃薯块茎萌芽的影响及其作用机制[J]. 作物学报, 2019, 45(2): 235-247. doi:10.3724/SP.J.1006.2019.84063
ZOU Xue, DING Fan, YU Jin-Long, PENG Jie, DENG Meng-Sheng, WANG Yu, LIU Li-Fang, YU-HAN Kai-Zong, CHEN Nian-Wei, WANG Xi-Yao.
马铃薯(Solanum tuberosum L.)适应性强、产量稳定、营养全面, 既可菜用又能加工成薯条、薯片、面食等。种植马铃薯是贫困地区脱贫增收的有效保证, 至2016年在中国的种植面积已达581.51万公顷, 总产9912.24万吨, 均列世界第一(FAO, 2016)。与水稻、小麦、玉米等籽粒型作物的种子相比, 块茎水分含量高, 贮藏期相对较短。种薯过早萌芽会造成薯块老化, 减产10%~30%[1]; 菜用或加工用, 薯块过早萌芽则消耗水分和营养物质, 降低商品价值。调控块茎萌芽, 减少贮藏损耗, 延长贮藏期对产业健康发展至关重要。
不同基因型的块茎休眠期各异, 休眠过后则萌芽, 2~5℃冷藏在一定时间内可延长休眠期但不能阻止萌芽, 常需配合使用抑芽剂来最大限度地延长贮藏期。目前用于加工、鲜食类块茎贮藏的抑芽剂主要是化学试剂氯苯胺灵(3-氯氨基甲酸异丙基酯, CIPC)。虽然CIPC属低毒类物质, 但随着人们对食品安全的重视, 市场可接受度在下降, 并且从1962年商业化到现在已有50多年, 它的降解物以及对环境的交叉污染引起越来越多的担忧[2,3]。从植物挥发性物质中寻找使用方便、无毒的绿色新型抑芽物质延长块茎贮藏, 是解决这一问题的理想方式。研究表明, 从一些芳香植物中提取的精油对马铃薯块茎萌芽具有不同程度的抑制作用。薄荷(Mentha haplocalyx L.)、芫荽(Coriandrum sativum L.)和尤加利(Myrtaceae eucalypteae L.)精油处理马铃薯块茎10 d后均能不同程度地延缓发芽, 但后两种精油中含有的柠檬烯会引起薯块大量腐烂[4]。另有研究表明留兰香(Mentha spicata L.)精油及其主要成分R-香芹酮能抑制马铃薯块茎发芽以及真菌和细菌活性[5,6,7]。1,4-二甲基萘是马铃薯休眠过程中自身产生的挥发性物质, 外源施用可抑制发芽, 但块茎表面残留量降低到1.4~2.7 mg kg-1时则会恢复发芽, 已证实该物质是通过提高细胞周期负控基因KRP1和KRP2的表达量, 阻碍细胞分裂, 从而抑制块茎发芽[8,9,10]。樟树叶中的主要成分樟脑(1,7,7-三甲基二环[2.2.1]庚烷-2-酮)对马铃薯块茎具有抑芽可逆效应, 分析块茎在休眠、萌芽、樟脑处理50 d及恢复萌芽4种状态下的转录组和蛋白组差异, 发现它并没有延长休眠状态, 而是使芽组织产生类似抵抗病原菌侵染的致死过程, 直接对芽造成损伤[11], 因处理时间过长, 并不清楚基因早期响应情况。
高通量测序技术(high-throughput mRNA sequencing, RNA-seq)是全面解析特定条件下基因表达变化的有效工具, 利用该技术共测得马铃薯休眠解除过程中的5912个差异表达基因, 其中生长素、赤霉素、细胞分裂素和油菜素内酯合成相关基因的过量表达占主导, 且伴随逆境响应和氧化还原调节[12]。同位素标记相对和绝对定量技术(isobaric tags for relative and absolute quantification, iTRAQ)是目前蛋白组学分析中应用效果较好的标记技术之一, 利用该方法从马铃薯块茎中鉴定出4453个蛋白, 在低温贮藏糖化过程中发现46个差异表达蛋白[13]。
本试验选用萘(1,4-二甲基萘的相似物)、樟脑、薄荷醇(2-异丙基-5-甲基环己醇, 薄荷精油主要成分) 3种挥发性物质处理马铃薯, 比较它们对块茎的抑芽效应; 同时, 用樟脑处理萌芽块茎3 d, 测定转录组和蛋白组, 挖掘早期响应的关键基因和蛋白; 根据组学分析, 结合基因表达量的变化幅度、基因的生物学意义和前人关于休眠和萌芽研究, 选择涉及细胞壁和表皮物质合成、植物病原菌互作等生理过程中的12个基因, 检测贮藏期间樟脑、薄荷醇处理对这些基因表达的影响。试验旨在确认具有潜在价值的抑芽物质, 初步认识其作用机制, 为马铃薯的绿色安全贮藏提供参考。
1 材料与方法
1.1 试验材料和试剂
马铃薯短休眠品种‘费乌瑞它’(Favorita)原原种, 由四川农业大学农学院马铃薯研究开发中心提供。2月底移栽试管苗于网室, 基质栽培, 于5月中下旬植株发黄时收获。萘购自成都科龙化工试剂厂, 樟脑(纯度96%)、薄荷醇(纯度98%)购自Sigma-Aldrich。TRIzol购自Invitrogen, 反转录试剂盒RevertAid First Strand cDNA Synthesis购自Thermo。引物及荧光染料套装SGExcel FastSYBR Mixture购自生工生物工程(上海)股份有限公司。
1.2 试验设计
挑选新收原原种(约7~10 g 粒-1), 常温下愈伤化14 d后以200 g L-1的量装入8 L密封硬纸盒。根据初筛结果, 设萘(Naphthalene, NAP)、樟脑(Camphor, CAM)、薄荷醇(Menthol, MEN) 3种试剂的浓度为0.2 g L-1, 分别称取固体颗粒用纱布包裹置盒中(不与薯块直接接触), 以不作任何处理为对照(CK)。每处理5盒, 于(23±2)℃室内贮藏。从贮藏0 d起, 每隔15~20 d观察统计其中3盒薯块发芽情况并称重, 芽长超过2 mm即认为该薯块已解除休眠而萌芽。调查每盒20个薯块的芽长, 统计时以1盒为整体计算平均芽长和标准差(n = 3)。贮藏0、7、21、35和49 d (此时CK的芽长多在2 mm)时, 从剩余2盒中分别取样冻存于-80℃用于1.4荧光定量检测, 樟脑和薄荷醇处理继续贮藏至70 d时取样。取样方式为, 以顶芽芽眼为中心, 用直径3 mm硬质塑料管打孔, 取从表皮起5 mm的圆柱体。1.3 RNA-seq和iTRAQ测序及分析
如图1所示, 贮藏到对照块茎芽长1~2 mm时, 用0.2 g L-1樟脑处理3 d后取样, 以顶芽芽眼为中心, 取直径3 mm, 高5 mm的圆柱体, 以8个圆柱体提取1管RNA或蛋白, 为1个生物学重复, 各3重复, 送深圳华大基因科技服务有限公司和杭州景杰生物科技有限公司完成RNA-seq和iTRAQ测序分析, 将3次重复的RNA或蛋白等量混合后建库测序。图1
新窗口打开|下载原图ZIP|生成PPT图1用作转录组和蛋白组测序的处理方式
Fig. 1Treatment schematics for the RNA-seq and iTRAQ experimental samples are listed
使用比对软件SOAPaligner/soap2将clean reads分别比对到马铃薯参考基因组和参考基因序列(http://potato.plantbiology.msu.edu/pgsc_download.shtml)[14], 以RPKM (Reads Per kb per Million reads)代表基因表达量, RPKM = (1,000,000×C)/(N×L×1000), 其中C为唯一比对到基因A的reads数, N为比对到所有参考基因的总reads数, L为基因A的碱基数[15]。使用Blastx将序列比对到KEGG、GO和NR数据库, 进行基因功能注释。对差异检验的P-value作多重假设检验校正, 通过控制 FDR (False Discovery Rate)来决定P-value的域值, 规定差异表达基因为倍数差异在2倍以上且FDR≤0.001。Gene Ontology功能显著性富集分析(GO分析)和Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway显著性富集分析同常规方法。规定蛋白丰度水平差异倍数大于1.3, 且经t检验其P值<0.05的蛋白为差异蛋白。在Gene Ontology数据库(http.//www.geneontology.org/)计算每个term的差异蛋白数目, 确定出差异蛋白显著富集的GO条目, Pathway的显著性分析同GO功能富集分析相似。将蛋白组和转录组作关联分析, 找出蛋白所关联到的基因, 即iTRAQ检测到的蛋白所对应的编码基因在RNA-seq水平也同样被检测到。设置差异蛋白和差异表达基因的筛选条件与前述相同, 进一步分析这些基因在iTRAQ和RNA-seq水平上的表达变化是否相同, 以及GO条目、代谢通路富集情况。
利用荧光定量验证测序结果准确性, 选择表1所示12个基因和SMO1、CYCD3、SN2等7个基因验证RNA-seq, 选择Transport protein Sec61、Heat shock protein 90-6、Protochlorophyllide reductase-like、TSI-1 protein、Glutathione S-transfera等5个蛋白的编码基因验证iTRAQ, 相关序列及引物参见文献[11], 基因序列http://solanaceae.plantbiology. msu.edu/cgi-bin/annotation_report.cgi参考文献[14]。RNA提取、荧光定量体系和计算方法参考文献[16]。
Table 1
表1
表1试验选择基因的引物序列
Table 1
| 基因 Gene | 编号 ID | 引物 Primers (5°-3°) | 长度 Length (bp) | 熔解温度 Melt temp. (℃) |
|---|---|---|---|---|
| 延伸因子EF1αL (内参Reference gene) Elongation factor 1 alpha-like | PGSC0003DMT400014674 | CTTGTACACCACGCTAAGGAG GTCAATGCAAACCATTCCTTG | 155 | 81.5 |
| 果胶裂解酶 PEL Pectate lyase | PGSC0003DMT400051938 | TGGGAAGGATCATGGAAACA ACCAGCATTTGAGGTAATCATT | 168 | 83.0 |
| 果胶酯酶PME Pectinesterase | PGSC0003DMT400005343 | AATCTACTGGTGTCGCTTAC TATTGTGGCATTGGTTCA | 167 | 78.5 |
| 多聚半乳糖醛酸酶PG Polygalacturonase | PGSC0003DMT400035162 | CAAGTAAAGGCTACGACAA TATGGACCTGAGAATGAAA | 199 | 81.0 |
| 细胞色素P450, CYP77A6 Cytochrome P450, CYP77A6 | PGSC0003DMT400060106 | CTTAGTCCACGAAGCATT TTCCGTAGTGACCTCCA | 141 | 84.0 |
| ω-羟基酸O-阿魏酸酯转移酶HPFT Omega-hydroxypalmitate O-feruloyl transferase | PGSC0003DMT400020180 | AGGTCACTTATCCGTTCC GAGTTGGCAATTTCAGG | 110 | 79.0 |
| 甘油二酯O-酰基转移酶WES Diacylglycerol O-acyltransferase | PGSC0003DMT400023554 | GTGTTGACCCGATTACCCA CTTGTTTAGTGACAGCCTCTT | 295 | 81.5 |
| AG-基序结合蛋白 GATA4L GATA transcription factor 4-like | PGSC0003DMT400030708 | GGTTGTTACTACGATGCTCT AATTATTGGATGTATCTTCCAC | 121 | 80.5 |
| Kip相关蛋白4 KRP4 Kip-related protein 4 | PGSC0003DMT400019919 | AAAAGACAGAATCGGAGTT GTCCTCTCTCTACCATCAAT | 182 | 81.0 |
| 氨基环丙烷羧酸氧化酶ACO Aminocyclopropanecarboxylate oxidase | PGSC0003DMT400030676 | CAGAGGAGCATCGTTACTGG AAAGAACCCTGCCTCCAAAC | 194 | 82.0 |
| 转录因子WRKY 75 WRKY transcription factor 75 | PGSC0003DMT400056352 | CAGCATCATCGTCGTCAT CATTAGTCCCAAGAACCC | 131 | 77.5 |
| 病程相关蛋白STH-2 Pathogenesis-related protein STH-2 | PGSC0003DMT400003934 | AAAACCAGGCATGGAACT ACGTGTAGACCTGATTCTTT | 99 | 79.0 |
| 呼吸爆发氧化酶(NADPH氧化酶) RBOH Respiratory burst oxidase (NADPH oxidase) | PGSC0003DMT400066027 | CTCTCATCACCATGCTCCAGG TGGCAGTGTCGTCTACCATAT | 168 | 82.5 |
新窗口打开|下载CSV
1.4 Real-time PCR检测贮藏期间基因表达变化
根据组学分析获得的差异基因和富集的代谢通路信息以及关于块茎萌芽的文献报道[10,11], 选择12个基因利用Oligo 6.0软件设计特异性引物(表1), RNA提取、荧光定量体系和计算方法同上。以贮藏0 d时的ΔCt值为对照, 将3次试验重复所提的3管RNA混合后反转录所得cDNA作3次荧光定量重复。2 结果与分析
2.1 抑芽能力对比
贮藏50 d时各处理差异明显, CK有70.90%长出约2 mm完整小芽, 萘处理长出分化不明显的畸形芽, 樟脑处理的芽顶端发黑坏死, 薄荷醇处理仅能见到内部死亡组织(图2)。80 d时CK的芽长已达14.29 mm, 而萘处理使芽生长受抑保持在2 mm左右, 樟脑处理的芽已全部死亡, 薄荷醇处理的薯块在整个贮藏期间均无芽生长(表2)。据此得出抑芽能力为薄荷醇>樟脑>萘。图2
新窗口打开|下载原图ZIP|生成PPT图2贮藏50 d时各处理的抑芽作用比较
CK: 对照; NAP: 萘处理; CAM: 樟脑处理; MEN: 薄荷醇处理。
Fig. 2Sprouting inhibition comparison of different treatments at 50 days
CK: control; NAP: naphthalene treatment; CAM: camphor treatment; MEN: menthol treatment.
Table 2
表2
表2贮藏期间各处理的平均芽长及块茎重量损失
Table 2
| 处理 Treatment | 芽长 Shoot length (mm) | 块茎重量损失 Tuber weight loss (%) | |||||
|---|---|---|---|---|---|---|---|
| 50 d | 65 d | 80 d | 60 d | 120 d | 180 d | ||
| CK | 2.45±0.36 aA | 6.02±0.37 A | 14.29±1.33 A | 6.68±0.36 aA | 13.85±0.61 A | 22.78±0.99 A | |
| NAP | 2.28±0.36 abA | 2.23±0.27 B | 2.06±0.47 B | 6.97±0.58 aA | 11.88±0.92 B | 17.49±0.93 B | |
| CAM | 1.59±0.45 bA | 1.31±0.20 C | 凋亡Death | 5.67±0.36 bAB | 9.42±0.67 C | 14.30±1.17 C | |
| MEN | 无芽生长 No bud growth | 4.83±0.27 cB | 7.37±0.55 D | 8.14±1.01 D | |||
新窗口打开|下载CSV
贮藏期间块茎的重量损失包括呼吸消耗、水分利用和散失等。休眠期间块茎重量损失小且各处理间差异不大, 随着对照萌芽和芽生长对营养物的消耗, 重量损失明显, 180 d时已达22.78% (表2)。3种物质对芽有不同程度的抑制作用, 减少了物质消耗, 使重量损失变缓。180 d时抑芽能力最强的是薄荷醇, 其损失率约只有对照的1/3, 具有延长块茎贮藏期的潜力。
2.2 差异表达基因和蛋白分析
由上述结果可知, 3种物质均对芽的生长具有直接抑制效应, 萘的作用较弱, 薄荷醇的致死效应则太强, 所以选择樟脑作进一步研究。芽长1~2 mm是块茎解除休眠萌芽的标志, 前期体视镜检表明直接处理萌芽块茎3 d造成顶端褐化, 说明芽开始受抑制, 试验检测此时的mRNA和蛋白变化, 以探寻樟脑抑芽效应早期在分子层面的影响。通过RNA-seq检测到23,553个基因, 其中有1227个基因表达显著上调, 299个显著下调; 用iTRAQ鉴定出2510个蛋白, 处理共造成296个蛋白显著上调, 204个显著下调。2.2.1 qRT-PCR验证测序结果 为验证RNA-seq测序结果的可靠性, 选择19个基因进行qRT-PCR, 由图3可知, 两种方式检测到基因表达变化趋势相似, 相关系数达0.9260**, 说明RNA-seq获得的基因表达信息具有较高的准确性和可信度。但qRT-PCR验证iTRAQ的蛋白表达则不一致, 有2个基因的表达变化趋势在转录和蛋白层面上出现相反情况(结果未列出)。推测蛋白合成、加工修饰滞后于mRNA转录, 两者在时间和空间上的差异造成变化趋势的不同步, qRT-PCR不适合用于验证iTRAQ。
图3
新窗口打开|下载原图ZIP|生成PPT图3荧光定量验证转录组测序
Fig. 3Quantitative RT-PCR validation of RNA-seq
2.2.2 差异表达基因和蛋白的GO、KEGG分析
Gene Ontology(GO)是一个国际标准化的基因功能分类体系, 分为生物过程(biological process, BP)、细胞组成(cellular component, CC)和分子功能(molecular function, MF)。将差异基因和蛋白通过GO富集分析, 在1526个差异表达基因中, 分别有712、622和780个被注释到BP、CC和MF条目中, 而差异蛋白则分别注释到289、129和361个。通过RNA-seq技术找出的差异基因编码蛋白显著富集的GO条目(P<0.05)表明, 差异蛋白主要在膜和细胞周围, 具有氧化还原酶活性、糖基转移酶活性、铁离子结合等功能, 主要参与响应刺激、胁迫、防御反应等过程。与转录组信息相比, 蛋白组可提供更下游的信息, 即直接起作用或受影响的蛋白。差异蛋白在细胞器内膜、线粒体内膜、染色体等多个部位均有分布。与转录组结果相似, 这些蛋白具有氧化还原酶活性、磷酸转移酶活性, 同样参与响应刺激、胁迫和防御反应。转录和蛋白水平都表明樟脑引起了芽响应刺激、胁迫和防御反应, 从分子水平上证明仅处理3 d即对芽产生了不利影响(图4)。
图4
新窗口打开|下载原图ZIP|生成PPT图4樟脑处理的差异表达基因和蛋白的显著GO富集
Fig. 4Significant GO terms analysis of differentially expressed genes and proteins in camphor treatment
通过KEGG分析, 差异表达基因共富集到113个代谢通路, 其中18个达显著水平(P<0.05), 表3列出了前10个通路。除次生代谢物生物合成外, 植物病原菌互作通路富集的基因最多, 其中的病程相关蛋白STH-2 、PR2和转录因子WRKY75在mRNA和蛋白水平都升高。谷胱甘肽代谢通路富集的24个基因中, 有 22个均编码谷胱甘肽S-转移酶并上调表达, 已知谷胱甘肽作为生物体内的一种重要抗氧化剂, 其主要作用是清除自由基, 保护蛋白质和酶等分子中的巯基, 相关酶的mRNA上调可以合成更多的抗氧化物质。二苯乙烯、黄酮类化合物等植物次生代谢产物具有抗氧化、抑菌等多种生理活性[17]。本试验中, 樟脑处理使上调表达基因显著富集到异黄酮、二苯乙烯类、黄酮和黄酮醇的生物合成通路, 相关基因如大豆异黄酮7-O-葡糖基转移酶基因、类黄酮3’-单加氧酶基因、反式白藜芦醇-di-O-甲基转移酶基因的表达均极显著升高。由此推测块茎首先通过促进合成不同种类的保护性物质, 缓解处理引起的活性氧紊乱来应对胁迫。
Table 3
表3
表3RNA-seq测序得到的差异基因富集到的前10个代谢通路
Table 3
| 代谢路径 Pathway | 注释到该路径的差异基因数 DEGs with pathway annotation | P值 P-value | 路径ID号 Pathway ID | |
|---|---|---|---|---|
| 谷胱甘肽代谢Glutathione metabolism | 23↑ | 1↓ | 5.32E-08 | ko00480 |
| 异黄酮生物合成Isoflavonoid biosynthesis | 15↑ | 0↓ | 4.61E-05 | ko00943 |
| 玉米素生物合成Zeatin biosynthesis | 26↑ | 2↓ | 5.88E-05 | ko00908 |
| 二苯乙烯类化合物、二芳基庚烷和姜辣素生物合成 Stilbenoid, diarylheptanoid and gingerol biosynthesis | 25↑ | 10↓ | 9.78E-05 | ko00945 |
| 柠檬烯和蒎烯降解Limonene and pinene degradation | 22↑ | 5↓ | 3.16E-04 | ko00903 |
| 植物病原菌互作Plant-pathogen interaction | 101↑ | 8↓ | 3.21E-04 | ko04626 |
| 内质网蛋白质加工 Protein processing in endoplasmic reticulum | 25↑ | 15↓ | 3.97E-04 | ko04141 |
| 黄酮和黄酮醇生物合成Flavone and flavonol biosynthesis | 21↑ | 1↓ | 9.68E-04 | ko00944 |
| 苯丙素生物合成Phenylpropanoid biosynthesis | 27↑ | 10↓ | 2.75E-03 | ko00940 |
| 次生代谢物的生物合成 Biosynthesis of secondary metabolites | 117↑ | 37↓ | 5.05E-03 | ko01110 |
新窗口打开|下载CSV
iTRAQ测得的差异蛋白显著富集到8个代谢通路, 核糖体(ko03010)、苯丙素生物合成(ko00940)、过氧化物酶体(ko04146)等与保护性次生物质合成、抗氧化相关通路中的蛋白上升, 而参与正常生理功能的泛素介导蛋白水解(ko04120)、内质网蛋白质加工(ko04141)等通路中的蛋白下降, 说明处理3 d已影响到正常的蛋白代谢和生理功能。
2.2.3 差异蛋白和mRNA关联分析 将iTRAQ检测到的2510个蛋白在转录组中进行关联分析, 其中458个差异蛋白对应的mRNA没有显著变化, 32个变化一致, 10个变化相反, 还有78个仅在mRNA水平变化, 也说明通过RNA-seq检测到的1526个显著表达变化的mRNA中, 仅有42个对应的蛋白变化, 其余蛋白则没有变化或没有检测到。
在两水平变化趋势相同的基因主要参与响应逆境、刺激、氧化胁迫、过氧化氢分解代谢、防御反应等生物过程, 发挥过氧化物酶活性、抗氧化活性、氧化还原活性等功能(图5)。关联基因的KEGG分析表明, 樟脑短期处理主要引起苯丙素生物合成(ko00940)通路中的多数基因表达上调, 相关蛋白升高。已知苯丙素生物合成的代谢产物包括木质素合成所需单体, 如香豆醇、松泊醇、5-羟基松柏醇、芥子醇, 而木质素是保护植物体的第一道防线表皮和细胞壁的重要组成物质。樟脑处理芽3 d使这些物质合成的酶表达量升高, 有利于表皮的合成和修复, 推测是前期抵抗外源侵害的重要响应方式之一。
图5
新窗口打开|下载原图ZIP|生成PPT图5转录组和蛋白组表达模式相同的基因GO富集
Fig. 5Correlation analyses in GO enrichment of genes with same expression trend between transcriptional and proteomics groups
2.3 贮藏期间抑芽物质对关键基因表达的影响
根据差异基因的变化幅度和主要富集的代谢通路信息, 选择参与保护性物质合成、生长发育、植物病原菌互作的基因12个, 检测它们在樟脑、薄荷醇处理下随贮藏时间的变化模式, 进一步明确两种抑芽物质的作用效应。2.3.1 参与细胞壁代谢、表皮物质合成基因的表达
果胶是植物细胞壁的主要构成物质, 果胶裂解酶(PEL)、果胶酯酶(PME)和多聚半乳糖醛酸酶(PG)参与果胶代谢, 从而导致胞壁降解, 细胞分离, 组织软化。已知果实成熟过程中这些基因的表达量和酶活性显著升高, 使果实软化。块茎贮藏过程中PEL、PME、PG表达量随贮藏时间升高, 并在萌芽时达到最高值, 分别为0 d时的94、85、133倍(图6), 说明块茎休眠解除过程伴随薯肉贮藏细胞的胞壁软化, 推测这更利于物质运输和营养供给。
图6
新窗口打开|下载原图ZIP|生成PPT图6抑芽物质对果胶代谢、角质、木栓和蜡质合成相关基因表达的影响
CK: 对照; CAM: 樟脑处理; MEN: 薄荷醇处理; CK-4℃: 对照4℃冷藏; 误差线为3个重复值的标准差。
Fig. 6Effects of sprouting-inhibitors on the expression of genes related to pectin metabolism and cutin, suberine, wax biosynthesis
CK: control; CAM: camphor treatment; MEN: menthol treatment; CK-4℃: control tuber storage at 4℃; the error line was the standard deviation of three repeated values.
樟脑处理下, 这3个基因的表达量在前期呈波动变化, 随着块茎解除休眠萌芽, 樟脑处理抑芽的同时使得这些基因的表达水平远低于同期对照, 49 d时只有同期对照的1.77%、18.19%和15.17%, 但都高于0 d的表达量。说明樟脑处理使芽的生长受抑, 对贮藏细胞的营养需求减少, 因此参与胞壁软化的基因表达量在贮藏中后期没有急剧上升。薄荷醇处理下, 除PME在21 d时升至对照同等水平外, 其余时期各基因的表达量均保持极低水平, 49 d时与仍处于休眠期的4℃贮藏块茎的表达量相似。
新生芽表皮角质层的形成对于防止水分散失和抵御外界胁迫至关重要, 角质层由角质、木栓、蜡质组成。CYP77A6、HPFT、WES基因参与形成角质、木栓和蜡质。由图6可知, 这3个基因在贮藏期间的变化趋势仍是升高, 特别在贮藏后期即休眠解除芽形成时。樟脑处理在早期引起这些基因表达量升高且高于同期对照, 7 d时HPFT和WES的表达量是同期对照的3.17倍和2.88倍, 但随时间增加, 这些基因的表达量没有继续升高, 而是保持不变或下降, 在35 d之后均低于同期对照。薄荷醇处理下, 除在7 d外整个贮藏期间都与0 d时的表达量相似或更低。推测樟脑、薄荷醇处理在早期不同程度地刺激这些基因的表达上调, 以促进保护层物质合成, 抵抗外界胁迫, 但随时间增加, 细胞结构受到破坏, 影响了这些物质合成, 继而加剧了芽死亡。
2.3.2 生长发育相关基因表达模式分析 由图7可知, 转录因子GATA4L的编码基因表达量随贮藏时间呈线性上升, 两者的相关系数达0.9450, 说明它与块茎休眠解除和萌芽密切相关。樟脑处理除35 d外其余时间点的表达量与0 d的相似, 而薄荷醇处理则更强烈地抑制该基因表达, 整个贮藏期间都只有0 d时的2%~8%。
图7
新窗口打开|下载原图ZIP|生成PPT图7抑芽物质对生长相关基因表达的影响
各处理的缩写同
Fig. 7Effects of sprouting-inhibitors on the expression of genes related to growth
The abbreviations of different treatments are the same as those given in
1-氨基环丙烷-1-羧酸氧化酶ACO是乙烯形成的关键酶, 随块茎休眠的解除ACO表达量升高, 到萌芽时为0 d的114倍(图7)。樟脑处理刺激该基因表达量提早升高, 7 d时达到同期对照的8.58倍, 但之后没有进一步上升, 随着对照表达量上升而低于同期对照, 到49 d时只有对照的1.65%。而无发芽迹象的薄荷醇处理中, ACO的表达量始终保持在低水平。
KRPs是细胞周期蛋白激酶抑制子, 负调控细胞分裂。尽管樟脑抑制萌芽, 但和对照相比, KRP4基因表达量在贮藏期间相近, 都呈下降趋势, 在49 d时的表达量均下降到0 d时的1/10。薄荷醇处理使该基因表达量显著升高, 在各时间点均高于同期对照(1.6~15.9倍)。随着细胞分裂负控基因KRP4的表达下降, 分生组织细胞分裂加快使休眠解除而萌芽, 推测薄荷醇通过提高KRP4的转录本, 起到阻碍细胞分裂抑制萌芽的作用。
2.3.3 植物病原菌互作相关基因表达模式分析
前述分析表明差异表达基因和蛋白参与防御反应, 选择植物病原菌互作通路中的3个基因, 分析它们在贮藏期间的表达变化特点。转录因子WRKY75传递MPK4信号, 诱导防御相关基因表达, WRKY75的表达在前期受樟脑和薄荷醇诱导, 21 d时分别升高至0 d的22.53倍和12.23倍, 但在21 d后薄荷醇处理的WRKY75表达量下降并一直保持低水平, 而樟脑处理的WRKY75表达量则在49 d时升高到0 d的100.55倍。STH-2属病程相关蛋白(PRP), 本试验中STH-2转录本在贮藏期间无明显变化, 但樟脑处理刺激该基因上调表达, 7 d 即达到0 d时的5.76倍, 在49 d块茎萌芽时的表达量增到0 d的343倍, 薄荷醇处理对该基因也有一定诱导作用但不如樟脑的明显。呼吸氧爆发氧化酶RBOH在互作通路中受钙调蛋白激酶激活, 产生活性氧, 引起过敏反应, 应答外界胁迫[18]。在贮藏中、后期RBOH表达量升高并伴随块茎休眠解除, 49 d萌芽时为0 d的6.87倍; 樟脑处理能刺激该基因上调表达, 在各时间点均高于同期对照, 49 d时达到最高值; 薄荷醇处理的最高值出现在第7天, 为0 d的17.76倍之后下调并保持较低水平(图8)。可以看出这3个基因在贮藏期间均受樟脑诱导表达, 并且都在块茎萌芽时达到最高值, 说明相对于休眠块茎, 这些基因更多地参与代谢更活跃的萌芽阶段对胁迫的响应, 随着芽死亡回到低水平。与樟脑不同的是, 薄荷醇能诱导RBOH基因在休眠期高表达, 说明休眠期的块茎可能受到薄荷醇较大的刺激作用。
图8
新窗口打开|下载原图ZIP|生成PPT图8抑芽物质对植物病原菌互作相关基因表达的影响
各处理的缩写同
Fig. 8Effects of sprouting-inhibitors on the expression of genes related to plant-pathogen interaction
The abbreviations of different treatments are the same as those given in
3 讨论
樟脑具有杀虫、消肿、止痛等多种医疗作用, 目前尚无在植物抑芽方面的研究; 薄荷醇可用作饮料和糖果等的赋香剂, 2003年首次发现它对离体芽有抑制作用, 但没有后续研究和应用[19]。本试验表明, 这两种物质都能抑制块茎萌芽, 而马铃薯自身的抑芽物质1,4-二甲基萘的相似物萘的抑芽能力则相对较弱。樟脑对块茎的抑芽效应可逆, 具有应用于种薯潜力; 薄荷醇的抑芽能力更强, 它本身也用于食品, 相对更安全, 具有用于商品薯贮藏的潜力。本试验采用自然挥发的处理方式, 所需试剂浓度较高, 下一步可通过雾化、熏蒸等方法, 增强试剂本身的附着效率来降低使用浓度以减少成本。樟脑长期处理薯块(50 d)主要引起与生长发育组织构成相关的基因下调表达[11]。本试验中, 樟脑处理芽3 d引起的上调基因居多, 它们具有合成保护性物质和抗氧化功能, 主要参与防御反应、响应逆境胁迫。上述对比表明, 在与抑芽物质接触早期, 芽组织积极应对胁迫, 而后期芽组织已受侵害甚至死亡, 生长发育相关基因表达下调。无论是休眠还是萌芽阶段, 植物病原菌互作通路中的病程相关蛋白STH2、PR2、转录因子WRKY75等的编码基因表达量都受樟脑诱导表达, 但萌芽阶段的表达量最高, 说明这些基因更多地参与代谢更活跃的萌芽阶段对胁迫的响应。关联分析除了找到蛋白与mRNA变化相同的32个基因, 多数基因表现出变化不同步现象, 在以qRT-PCR验证蛋白测序结果时也发现两者变化不同步, 这与马进等[20]以qRT-PCR验证iTRAQ不符的情况相同。拟南芥的天冬氨酸蛋白酶猪笼草蛋白酶基因在不同组织如叶片、茎、种子和豆荚均有表达, 认为这类蛋白涉及多种功能如细胞程序性死亡、蛋白前体加工、病害和逆境抗性、叶片衰老等[21]。本试验中天冬氨酸蛋白酶猪笼草蛋白酶1转录下调而蛋白升高, 推测上述现象是转录和翻译在时间上不同步, 蛋白后期加工修饰造成了其变化滞后于转录应答, 使得mRNA变化已趋于平稳或下调后, 蛋白水平才上调以抵抗外界胁迫。
乙烯是植物的内源激素之一, 在调节衰老、果实成熟以及器官脱落等过程中发挥促进作用[22]。Coleman和Dioxide [23]研究表明外源乙烯能降低块茎的脱落酸含量, 促进休眠的解除, Hartmann等[24]研究证实休眠块茎分生组织活化与生长素及乙烯信号转导、细胞周期调控等的转录变化密切相关。将乙烯和典型的催芽剂赤霉素共同处理马铃薯促进长休眠品种萌芽[25]。本试验证实块茎贮藏过程伴随乙烯合成关键酶基因ACO表达量的百倍增加, 从mRNA水平验证乙烯合成是块茎解除休眠所经生理过程。乙烯可启动果实代谢发生质的变化, 如淀粉水解为糖、多聚半乳糖醛酸酶含量上升引起的组织软化、香气成分的合成等, 是果实成熟的重要启动因子。用乙烯处理芒果、玫瑰花等均观察到PEL、PME基因转录本增加和酶活性升高及果胶增溶现象[26,27]。马铃薯块茎休眠解除过程与果实成熟有相似之处, 两者都涉及淀粉的降解, 贮藏细胞胞壁的软化和分解等。本试验中乙烯合成关键酶ACO和果胶降解酶PEL、PME、PG表达量都随着贮藏时间而升高, 推测块茎休眠解除过程中合成乙烯并促进胞壁降解相关酶的基因表达, 加快营养物质运输, 这可能也是贮藏后期薯肉软化的原因之一。樟脑和薄荷醇处理抑制了这些基因的表达使块茎细胞壁在贮藏期间保持更好地完整性, 有效降低水分散失和保持薯块硬度。
StGATA4L属GATA转录因子家族, 已知拟南芥GATA家族成员有29个, 参与种子萌发、下胚轴伸长、叶序和花器官起始、开花和衰老等生理功能[28]。StGATA4L基因与拟南芥GATA家族中的AtGATA2、AtGATA4相似性最高, 但它们编码的氨基酸序列相似性也仅有50%。AtGATA2则能介导油菜素内酯和光信号的相互交流, 参与形态建成[29]。通过融合抑制子基因沉默技术抑制AtGATA4下游靶基因转录, 转基因拟南芥在缺氮胁迫下有更高的根毛密度和健壮的地上部, 认为AtGATA4参与根系发育[30]。本试验中StGATA4L表达量与块茎生理状态的转变密切相关, 它在块茎萌芽中的作用以及在功能上是否与拟南芥有相似性还需要进一步研究。KRPs通过抑制活化的CYCD-CDKA复合物, 阻碍细胞分裂由G1期向S期的过渡 [31]。提高KRPs表达会造成生长缓慢、器官变小、结实下降; 反之则植株器官、种子变大[32,33]。Campbell等[10]利用芯片和定量RT-PCR证实马铃薯产生的挥发性抑芽物1,4-二甲基萘是通过增加KRP1和KRP2的转录本阻碍细胞分裂达到抑芽效果的。本试验中薄荷醇处理同样提高了KRP4的转录本, 并表现出较强抑芽作用, 推测两者有相似的作用机制。
低浓度晚疫病菌孢子侵染时马铃薯STH-2蛋白在抗病品种中的升高速度快于感病品种, 积累量也高于后者, 将其归为病程相关蛋白PRP, 但通过转基因技术过量表达该基因并不能增强植株的晚疫病抗性, 但可提高马铃薯细胞对盐和渗透胁迫的耐性[34,35,36]。最近的研究表明青枯病菌侵染时, STH-2蛋白在抗病品种中大量积累[37]。上述研究说明STH-2参与马铃薯生物和非生物胁迫反应, 但具体功能和作用方式仍不清楚。本试验中, 樟脑处理不仅引起萌芽块茎STH-2转录本和蛋白水平极大的升高, 同时植物病原菌互作通路中的病程相关蛋白PR2、转录因子WRKY75等108个基因转录水平也升高, 推测该处理引起块茎产生与抵抗病菌侵染相似的应答。
通过上述分析, 认为樟脑处理首先引起防御反应, 使角质、蜡质等表皮保护层物质和黄酮、谷胱苷肽等抗菌、抗氧化物质合成相关基因的表达上调, 抵抗逆境胁迫; 随着樟脑处理伤害的加剧, 这些物质合成基因的表达下降, 与生长发育相关基因的表达受抑, 但植物病原菌互作通路中的各基因表达量进一步升高, 引起与抵抗病原菌侵染相似的应激反应, 芽逐渐坏死(图9)。薄荷醇处理的变化模式较为单一, 在整个贮藏期间表达量都较低, 除了引起KRP4和RBOH基因的表达量呈现出不同于樟脑处理的升高, 说明它和樟脑的抑芽机制不同。推测它对细胞周期蛋白的抑制作用是阻止块茎萌芽的重要原因, 而涉及的其他响应基因和生理过程, 还需进一步研究。
图9
新窗口打开|下载原图ZIP|生成PPT图9樟脑作用于块茎引起的响应模式推测
实线箭头: 处理与同期对照相比上调; 虚线箭头: 处理与同期对照相比下调, 箭头粗细代表变化程度。
Fig. 9Acting pattern speculation of tuber response to camphor treatment
Solid arrows: expression levels up-regulated compared with control; dotted arrows: expression levels down-regulated compared with control, the size of the arrow represented the degree of variability.
4 结论
樟脑和薄荷醇均能抑制生长发育相关基因的表达, 使萌发的芽死亡, 降低贮藏损耗。樟脑处理早期引发防御反应, 上调表达参与角质、抗氧化、抗菌等保护性物质合成的基因; 萌芽时造成与抵抗病原菌相似的强烈互作。薄荷醇提高KRP4表达阻碍细胞分裂是分生组织坏死的原因之一。参考文献 原文顺序
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测定云南马铃薯主栽品种'合作88'不同生理年龄种薯芽中内源激素含量的 结果表明,随着马铃薯种薯生理年龄的增加,各种内源激素含量均呈下降趋势,生理年龄30 d以后的种薯中,IAA、GA3、玉米素(ZR)和双氢玉米素(DHZR)下降显著,ABA下降相对缓慢;生理年龄30 d的种薯,主茎长出的叶片数多,单株薯重高.
URL [本文引用: 1]
测定云南马铃薯主栽品种'合作88'不同生理年龄种薯芽中内源激素含量的 结果表明,随着马铃薯种薯生理年龄的增加,各种内源激素含量均呈下降趋势,生理年龄30 d以后的种薯中,IAA、GA3、玉米素(ZR)和双氢玉米素(DHZR)下降显著,ABA下降相对缓慢;生理年龄30 d的种薯,主茎长出的叶片数多,单株薯重高.
DOI:10.1007/s11540-011-9188-zURL [本文引用: 1]
Marketplace preference for lower pesticide residues in foods has led to research to reduce the residue of chlorpropham (isopropyl N -3-chlorophenyl carbamate; CIPC), a postharvest-applied sprout inhibitor which is widely used around the globe to prevent sprouting of stored potatoes ( Solanum tuberosum L.). Ethylene gas, an effective, safe and non-toxic sprout inhibitor used in several countries, sometimes has negative effects on the colour of processed potato products when used alone. Trials were conducted over 302years using cv. Shepody (French fry) and cv. NorValley (potato chips/crisps) to determine whether a combination of these two sprout inhibitors, at reduced dosages, could inhibit sprouting while maintaining good processing colour. CIPC applied at 0, 0.1, 0.25 and 1.0 times the recommended dosage was combined with 402μll 611 of ethylene gas applied or no ethylene at all (0 ethylene), for 102day in 4 days, for 102day in 2 days or continuously, in a factorial design. Sprout inhibition in both cultivars was excellent at all levels of CIPC application except the 0 rate. In both cultivars, sprouting was inhibited by the continuous ethylene treatment. However, all levels of ethylene exposure except the 0 rate negatively affected processing colour in both cultivars. The darkening was dose dependent, whereby the colour was darkest in continuous ethylene and was less affected by the intermittent exposures. In continuous ethylene, the colour was progressively lighter during storage after initial darkening. Shepody tubers appeared to be more sensitive to ethylene than the NorValley tubers. In Shepody only, colour in the ethylene of 102day in 4 days treatments was progressively darker with increasing time in storage.
DOI:10.3920/QAS2017.1088URL [本文引用: 1]
DOI:10.1016/j.postharvbio.2013.02.017URL [本文引用: 1]
The replacement of synthetic chemical compounds Used as potato sprout inhibitors with naturally occurring compounds has been studied in potato varieties used in the processing industry. Sprout suppressant capacity of caraway (Carum carvi, L.), peppermint (Mentha piperita, L.), coriander (Coriandrum sativum, L) and eucalyptus (Eucalyptus globulus, Labill.) essential oils was studied and compared to the sprout suppressant capacity of chlorpropham (CIPC). For the particular conditions of this study, which used high concentrations, peppermint and coriander essential oils were the most effective sprout inhibitors with inhibition rates of more from 65 to 95% respect to their control. The use of these essential oils also prevented phytopathogenic damage. Analytical quality parameters, such as moisture content and total soluble solids, were not affected by treatment with any of the aromatic essential oils. Sensory analysis revealed no difference in appearance and taste between potatoes treated with essential oils and untreated tubers. (C) 2013 Elsevier B.V. All rights reserved.
DOI:10.1016/0926-6690(95)00005-WURL [本文引用: 1]
Carvone was recently launched as a sprout inhibitor for potatoes in the Netherlands under the trade name “Talent”.
DOI:10.1016/0926-6690(95)00007-YURL [本文引用: 1]
S-Carvone, a common monoterpene found in caraway (Carum carvi L.), inhibits the sprouting of potatoes very efficiently at continuous low head space concentrations. The length growth of potato sprouts was inhibited within 2 days following exposure to S-carvone. Sprouts were able to convert S-carvone into more reduced compounds. In addition, growth of the plant-pathogenic fungi Fusarium solani and Fusarium sulphureum was found to be inhibited by S-carvone at concentrations between 1 and 3 mM. At sub-inhibitory concentration, both F. solani and F. sulphureum converted S-carvone into more reduced compounds which were shown to be less toxic as compared to S-carvone. Finally, the growth rate of Streptococcus thermophilus, Lactococcus lactis and Escherichia coli decreased at S-carvone concentrations above 1 mM. This decrease correlated with a dissipation of the proton motive force generated in these cells.
DOI:10.1007/s00425-010-1154-5URLPMID:20390295 [本文引用: 1]
Sprouting of potatoes during storage, due to tuber dormancy release, is associated with weight loss and softening. Sprout-preventing chemicals, such as chlorpropham (CIPC), can negatively impact the environment and human health. Monthly thermal fogging with mint (Mentha spicata L.) essential oil (MEO) inhibited sprouting in eight potato cultivars during large-volume 6-month storage: the tubers remained firm with 38% lower weight loss after 140 days of storage. The sprout-inhibitory action may be nullified: treated tubers washed with water resumed sprouting within days, with reduced apical dominance. MEO application caused local necrosis of the bud meristem, and a few weeks later, axillary bud (AX) growth was induced in the same sprouting eye. MEO components analysis showed that 73% of its content is the monoterpene R-carvone. Tubers treated with synthetic R-carvone in equivalent dose, 4.5 l l-1, showed an inhibitory effect similar to that of MEO. Surprisingly, 0.5 l l-1 of MEO or synthetic R-carvone catalyzed AX sprouting in the tuber. To the best of our knowledge, this is the first report of an essential oil vapor inducing early sprouting of potato tubers. R-carvone caused visible damage to the meristem membrane at sprout-inhibiting, but not sprout-inducing doses, suggesting different underlying mechanisms. After 5 days' exposure to R-carvone, its derivatives transcarveol and neo-dihydrocarveol were found in buds of tubers treated with the inhibitory dose, suggesting biodegradation. These experiments demonstrate the potential of MEO vapor as an environmentally friendly alternative to CIPC in stored potatoes and as a research tool for the control of sprouting in plants.
DOI:10.1007/s12230-010-9146-3URL [本文引用: 1]
Research on new sprout control methods led to the discovery of 1,4dimethylnaphthalene (1,4DMN) in the late 1970s. This compound has been registered in the USA since 1996 as a reduced risk chemical for use on stored potatoes for the purpose of sprout suppression. Relatively little is known about the levels of tuber residues required to maintain sprout suppression. The objective of this study was to identify the minimum 1,4DMN tuber residue levels above which sprout growth was inhibited. Using modified steel drums to store and treat potatoes, rates of 00208L02kg 611 to 560208L02kg 611 1,4DMN were applied to non-dormant, slightly “peeping” seed tubers of four different cultivars (Russet Burbank, Russet Norkotah, Shepody and FL1879) stored at 15 C65±652C. Tubers were treated by dribbling 1,4DMN onto a gauze that was placed on top of a return air pipe that re-circulated forced air inside the drum. Higher application rates resulted in higher 1,4DMN residues and longer suppression of sprout growth. Sprout growth was controlled for more than 5002days for all cultivars in both years for tubers treated at the highest rate. The duration of sprout inhibition corresponded with the time it took for tuber residues to drop below an estimated residue level of 1.4–2.102ppm, for R. Burbank, Shepody and FL1879, and 2.702ppm for Russet Norkotah. Below these levels, sprouting was no longer suppressed and resumed for all cultivars.
DOI:10.1007/s11103-010-9607-6URLPMID:20135197 [本文引用: 1]
http://link.springer.com/article/10.1007%2Fs11103-010-9607-6
DOI:10.1007/s10142-011-0257-9URLPMID:22113341 [本文引用: 3]
The suppression of sprout growth is critical for the long-term storage of potato tubers. 1,4-Dimethylenapthlene (DMN) is a new class of sprout control agent but the metabolic mode of action for this compound has yet to be elucidated. Changes in transcriptional profiles of meristems isolated from potato tubers treated with the DMN were investigated using an Agilent 44 K 60-mer-oligo microarray. RNA was isolated from nondormant Russet Burbank meristems isolated from tubers treated with DMN for 3 days or activated charcoal as a control. RNA was used to develop probes that were hybridized against a microarray developed by the Potato Oligo Chip Initiative. Analysis of the array data was conducted in two stages: total array data was examined using a linear model and the software Limma and pathway analysis was conducted by linking the potato sequences to the Arabidopsis thaliana . DMN elicited a change in a number of transcripts associated with cold responses, water regulation, salt stress, and osmotic adjustment. DMN also resulted in a repression of cyclin or cyclin-like transcripts. DMN also resulted in a 50% decrease in thymidine incorporation suggesting a repression of the S phase of the cell cycle. Quantitative real-time polymerase chain reaction analysis demonstrated that DMN increased transcripts for the cell cycle inhibitors KRP1 and KRP2. We conclude the DMN results in alteration of genes associated with the maintenance of a G1/S phase block possibly through the induction of the cell cycle inhibitors KRP1 and KRP2.
DOI:10.3390/ijms18112280URLPMID:29084178 [本文引用: 4]
Sprouting regulation in potato tubers is important for improving commercial value and producing new plants. Camphor shows flexible inhibition of tuber sprouting and prolongs the storage period of potato, but its underlying mechanism remains unknown. The results of the present study suggest that camphor inhibition caused bud growth deformities and necrosis, but after moving to more ventilated conditions, new sprouts grew from the bud eye of the tuber. Subsequently, the sucrose and fructose contents as well as polyphenol oxidase (PPO) activity were assessed after camphor inhibition. Transcription and proteomics data from dormancy (D), sprouting (S), camphor inhibition (C), and recovery sprouting (R) samples showed changes in the expression levels of approximately 4000 transcripts, and 700 proteins showed different abundances. KEGG (Kyoto encyclopaedia of genes and genomes) pathway analysis of the transcription levels indicated that phytohormone synthesis and signal transduction play important roles in tuber sprouting. Camphor inhibited these processes, particularly for gibberellic acid, brassinosteroids, and ethylene, leading to dysregulation of physiological processes such as cutin, suberine and wax biosynthesis, fatty acid elongation, phenylpropanoid biosynthesis, and starch and sucrose metabolism, resulting in bud necrosis and prolonged storage periods. The KEGG pathway correlation between transcripts and proteins revealed that terpenoid backbone biosynthesis and plant 損athogen interaction pathways showed significant differences in D vs. S samples, but 13 pathways were remarkably different in the D vs. C groups, as camphor inhibition significantly increased both the transcription levels and protein abundance of pathogenesis-related protein PR-10a (or STH-2), the pathogenesis-related P2-like precursor protein, and the kirola-like protein as compared to sprouting. In recovery sprouting, these genes and proteins were decreased at both the transcriptional level and in protein abundance. It was important to find that the inhibitory effect of camphor on potato tuber sprout was reversible, revealing the action mechanism was similar to resistance to pathogen infection. The present study provides a theoretical basis for the application of camphor in prolonging seed potato storage.
DOI:10.1016/j.jbiotec.2015.01.019URLPMID:25661840 [本文引用: 1]
Potato tuber dormancy release is a critical development process that allows potato to produce new plant. The first Illumina RNA sequencing to generate the expressed mRNAs at dormancy tuber (DT), dormancy release tuber (DRT) and sprouting tuber (ST) was performed. We identified 26,639 genes including 5,912 (3,450 up-regulated while 2,462 down-regulated) and 3,885 (2,141 up-regulated while 1,744 down-regulated) genes were differentially expressed from DT vs DRT and DRT vs ST. The RNA-Seq results were further verified using qRT-PCR. We found reserve mobilization events were activated before the bud emergence (DT vs DRT) and highlighted after dormancy release (DRT vs ST). Overexpressed genes related to metabolism of auxin, gibberellic acid, cytokinin and barssinosteriod were dominated in DT vs DRT, whereas overexpressed genes involved in metabolism of ethylene, jasmonate and salicylate were prominent in DRT vs ST. Various histone and cyclin isoforms associated genes involved in cell division/cycle were mainly up-regulated in DT vs DRT. Dormancy release process was also companied by stress response and redox regulation, those genes related to biotic stress, cell wall and second metabolism was preferentially overexpressed in DRT vs ST, which might accelerate dormancy breaking and sprout outgrowth. The metabolic processes activated during tuber dormancy release were also supported by plant seed models. These results represented the first comprehensive picture of a large number of genes involved in tuber dormancy release process.
DOI:10.1021/pr200455sURLPMID:21842911 [本文引用: 1]
Abstract Cold-induced sweetening in potato tubers is a costly problem for the food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 months of storage at 5 C. We evaluated both high pH reverse phase (hpRP) liquid chromatography (LC) and off-gel electrophoresis (OGE) as first dimension fractionation methods followed by nanoLC-MS/MS, using two high performance mass spectrometry platforms (Q-TOF and Orbitrap). We found that hpRP-LC consistently offered better resolution, reduced expression ratio compression, and a more MS-compatible workflow than OGE and consistently yielded more unique peptide/protein identifications and higher sequence coverage with better quantification. In this study, a total of 4463 potato proteins were identified, of which 46 showed differential expressions during potato tuber cold storage. Several key proteins important in controlling starch-sugar conversion, which leads to cold-induced sweetening, as well as other proteins that are potentially involved in this process, were identified. Our results suggest that the hpRP-RP shotgun approach is a feasible and practical workflow for discovering potential protein candidates in plant proteomic analysis.
DOI:10.1038/nature10158URLPMID:21743474 [本文引用: 2]
Abstract Potato (Solanum tuberosum L.) is the world's most important non-grain food crop and is central to global food security. It is clonally propagated, highly heterozygous, autotetraploid, and suffers acute inbreeding depression. Here we use a homozygous doubled-monoploid potato clone to sequence and assemble 86% of the 844-megabase genome. We predict 39,031 protein-coding genes and present evidence for at least two genome duplication events indicative of a palaeopolyploid origin. As the first genome sequence of an asterid, the potato genome reveals 2,642 genes specific to this large angiosperm clade. We also sequenced a heterozygous diploid clone and show that gene presence/absence variants and other potentially deleterious mutations occur frequently and are a likely cause of inbreeding depression. Gene family expansion, tissue-specific expression and recruitment of genes to new pathways contributed to the evolution of tuber development. The potato genome sequence provides a platform for genetic improvement of this vital crop. 2011 Macmillan Publishers Limited. All rights reserved
DOI:10.1038/nmeth.1226URL [本文引用: 1]
DOI:10.3724/SP.J.1006.2017.00811URL [本文引用: 1]
为探明油菜素内酯BR在块茎萌芽中的作用,建立更有效的种薯催芽调控体系,选择了休眠期不同的3个品种,利用q RT-PCR分析与BR合成、信号转导、调控有关的9个基因在贮藏期间及抑芽处理下的表达模式;同时检测BR类似物24-表油菜素内酯(24-e BL)及其与赤霉素GA3对块茎萌芽的影响。结果表明,涉及BR合成的4个基因表达量均随贮藏时间延长升高,短休眠品种升高的时间点早于中、长休眠品种;信号转导及调控基因中BRI1和CYCD3的变化与合成基因相似,BSK和TCH4的表达量则在中、长休眠期品种中保持恒定。抑芽处理在贮藏前期能刺激这些基因的表达升高,但之后都迅速下降并保持低水平。转录因子BZR1在各品种中以及抑芽处理下均没有明显变化。24-e BL利于块茎解除休眠,但不促进芽的伸长生长,与GA3互配效果更佳,单株块茎增重37.92%~98.41%。结论表明,BR合成和信号转导是块茎从休眠向萌芽转变的必经生理过程,它与GA3互配用于催芽更利于种薯萌芽的整齐、健壮并促进块茎形成。
DOI:10.3724/SP.J.1006.2017.00811URL [本文引用: 1]
为探明油菜素内酯BR在块茎萌芽中的作用,建立更有效的种薯催芽调控体系,选择了休眠期不同的3个品种,利用q RT-PCR分析与BR合成、信号转导、调控有关的9个基因在贮藏期间及抑芽处理下的表达模式;同时检测BR类似物24-表油菜素内酯(24-e BL)及其与赤霉素GA3对块茎萌芽的影响。结果表明,涉及BR合成的4个基因表达量均随贮藏时间延长升高,短休眠品种升高的时间点早于中、长休眠品种;信号转导及调控基因中BRI1和CYCD3的变化与合成基因相似,BSK和TCH4的表达量则在中、长休眠期品种中保持恒定。抑芽处理在贮藏前期能刺激这些基因的表达升高,但之后都迅速下降并保持低水平。转录因子BZR1在各品种中以及抑芽处理下均没有明显变化。24-e BL利于块茎解除休眠,但不促进芽的伸长生长,与GA3互配效果更佳,单株块茎增重37.92%~98.41%。结论表明,BR合成和信号转导是块茎从休眠向萌芽转变的必经生理过程,它与GA3互配用于催芽更利于种薯萌芽的整齐、健壮并促进块茎形成。
DOI:10.1038/35081178URL [本文引用: 1]
DOI:10.1105/tpc.106.048884URLPMID:17400895 [本文引用: 1]
Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by$\text{Ca}^{2+}$. However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.
DOI:10.1007/BF02736098URL [本文引用: 1]
Single or combined extracts of black thyme, fennel, sage, wild tea and wild mint were used to evaluate in vitro antibacterial activity against common pathogenic bacteria and lactic acid bacteria (LAB). The antibacterial effects of combined plant extracts (1 to 1 mixing ratio) were similar to single plant extracts against pathogenic bacteria. The inhibition of LAB was weaker with single plant... [Show full abstract]
DOI:10.3969/j.issn.1674-7968.2016.04.004URL [本文引用: 1]
为了从蛋白质水平上揭示南方型紫花苜蓿（Medicago sativa＇Millennium＇）适应盐胁迫的分子机制,本研究以30 d苗龄的南方型紫花苜蓿实生苗为材料,分析正常培养和250 mmol/L Na Cl处理72 h后根中的蛋白表达变化,采用同位素相对标记与绝对定量技术（isobaric tags for relative and absolute quantitation,i TRAQ）结合双向液相色谱与串联质谱（2-dimensional liquid chromatography-tandem mass spectrometry,2D-LC-MS/MS）定量蛋白质组技术鉴定南方型紫花苜蓿根部响应盐胁迫差异表达蛋白,对所获得差异蛋白进行生物信息学分析,筛选出可能耐盐潜在靶标蛋白。同时,选择5个差异表达蛋白进行实时荧光定量PCR（q RT-PCR）验证。结果表明,共鉴定3 857种定量蛋白,534种差异蛋白（变化倍数≥1.2,P〈0.05）,其中表达上调的281种,表达下调的253种。基因本体（Gene Ontology,GO）分子功能提示这些差异性蛋白主要参与转运活性、催化活性和酶调控活性。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）通路显著性富集于代谢途径、次生代谢产物生物合成、苯丙氨酸代谢和核糖体等（P〈0.05,错误发现率（false discovery rate,FDR）〈0.05）。成功鉴定的差异蛋白分别涉及到信号传递（8.99%）、抗氧化物（7.68%）、防御（5.61%）、蛋白质合成、加工和降解（14.79%）、能量产生与转运（5.81%）、代谢（26.97%）、膜与胞内运输（5.62%）和细胞结构、分裂和细胞骨架（2.06%）等。差异表达蛋白中与信号传递、抗氧化物和防御等相关的蛋白表达量总体上调,而与代谢和能量产生与转运相关蛋白表达量总体下调。q RT-PCR实验发现,m RNA与蛋白质表达水平并不一致。研究发现组氨酸磷酸转移蛋白、丝裂原活化蛋白激酶、h类硫氧还蛋白、蛋氨酸亚砜还原酶基因、鸟苷二磷酸（guanosine diphosphate,GDP）解离抑制因子、脂质17
DOI:10.3969/j.issn.1674-7968.2016.04.004URL [本文引用: 1]
为了从蛋白质水平上揭示南方型紫花苜蓿（Medicago sativa＇Millennium＇）适应盐胁迫的分子机制,本研究以30 d苗龄的南方型紫花苜蓿实生苗为材料,分析正常培养和250 mmol/L Na Cl处理72 h后根中的蛋白表达变化,采用同位素相对标记与绝对定量技术（isobaric tags for relative and absolute quantitation,i TRAQ）结合双向液相色谱与串联质谱（2-dimensional liquid chromatography-tandem mass spectrometry,2D-LC-MS/MS）定量蛋白质组技术鉴定南方型紫花苜蓿根部响应盐胁迫差异表达蛋白,对所获得差异蛋白进行生物信息学分析,筛选出可能耐盐潜在靶标蛋白。同时,选择5个差异表达蛋白进行实时荧光定量PCR（q RT-PCR）验证。结果表明,共鉴定3 857种定量蛋白,534种差异蛋白（变化倍数≥1.2,P〈0.05）,其中表达上调的281种,表达下调的253种。基因本体（Gene Ontology,GO）分子功能提示这些差异性蛋白主要参与转运活性、催化活性和酶调控活性。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes,KEGG）通路显著性富集于代谢途径、次生代谢产物生物合成、苯丙氨酸代谢和核糖体等（P〈0.05,错误发现率（false discovery rate,FDR）〈0.05）。成功鉴定的差异蛋白分别涉及到信号传递（8.99%）、抗氧化物（7.68%）、防御（5.61%）、蛋白质合成、加工和降解（14.79%）、能量产生与转运（5.81%）、代谢（26.97%）、膜与胞内运输（5.62%）和细胞结构、分裂和细胞骨架（2.06%）等。差异表达蛋白中与信号传递、抗氧化物和防御等相关的蛋白表达量总体上调,而与代谢和能量产生与转运相关蛋白表达量总体下调。q RT-PCR实验发现,m RNA与蛋白质表达水平并不一致。研究发现组氨酸磷酸转移蛋白、丝裂原活化蛋白激酶、h类硫氧还蛋白、蛋氨酸亚砜还原酶基因、鸟苷二磷酸（guanosine diphosphate,GDP）解离抑制因子、脂质17
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DOI:10.1006/anbo.1998.0645URL [本文引用: 1]
Potato cv. Russet Burbank tubers which had been stored at 3 or 13deg C were exposed for 1-7 days during early or late dormancy to ambient air or the following gas compositions: (I) 60% CO2 + 20% O2 + 20% N2; (II) 20% CO2 + 40% O2 + 40% N2. In the earlyphase, gas II was more effective in breaking dormancy than gas I in tubers stored at 3deg C, while gas II was not effective in tubers stored at 13deg C. In the late phase, effects of gases were generally smaller, with gas I being more effective. Sprout growthwas greatest in tubers stored at 3deg C treated with gas I. Both gas I and ethylene decreased ABA levels in the apical eye region of tubers within 24 h. Gas II reduced ABA levels to a lesser extent, while the effects of both gases were increased by ethylene.
DOI:10.1104/pp.110.168252URLPMID:21163959 [本文引用: 1]
Abstract Reactivation of dormant meristems is of central importance for plant fitness and survival. Due to their large meristem size, potato (Solanum tuberosum) tubers serve as a model system to study the underlying molecular processes. The phytohormones cytokinins (CK) and gibberellins (GA) play important roles in releasing potato tuber dormancy and promoting sprouting, but their mode of action in these processes is still obscure. Here, we established an in vitro assay using excised tuber buds to study the dormancy-releasing capacity of GA and CK and show that application of gibberellic acid (GA(3)) is sufficient to induce sprouting. In contrast, treatment with 6-benzylaminopurine induced bud break but did not support further sprout growth unless GA(3) was administered additionally. Transgenic potato plants expressing Arabidopsis (Arabidopsis thaliana) GA 20-oxidase or GA 2-oxidase to modify endogenous GA levels showed the expected phenotypical changes as well as slight effects on tuber sprouting. The isopentenyltransferase (IPT) from Agrobacterium tumefaciens and the Arabidopsis cytokinin oxidase/dehydrogenase1 (CKX) were exploited to modify the amounts of CK in transgenic potato plants. IPT expression promoted earlier sprouting in vitro. Strikingly, CKX-expressing tubers exhibited a prolonged dormancy period and did not respond to GA(3). This supports an essential role of CK in terminating tuber dormancy and indicates that GA is not sufficient to break dormancy in the absence of CK. GA(3)-treated wild-type and CKX-expressing tuber buds were subjected to a transcriptome analysis that revealed transcriptional changes in several functional groups, including cell wall metabolism, cell cycle, and auxin and ethylene signaling, denoting events associated with the reactivation of dormant meristems.
DOI:10.1007/s12230-010-9178-8URL [本文引用: 1]
While greenhouse production of minitubers has markedly improved production of disease free seed, minituber dormancy can be an issue. This study tested application of abscisic acid (ABA), ethylene (E), gibberellic acid (GA) and the combination of E+GA on release of dormancy in potato minitubers, sprout development, and subsequent effects on field plant growth and yield of seed tubers. Cultivars were categorized on the basis of known dormancy responses as follows: (1) one short dormancy cultivar, Silverton Russet, (2) three intermediate dormancy cultivars, Russet Norkotah (RNK), Sangre S-14 and Desiree, and (3) two long dormancy cultivars, Nooksack and Russet Norkotah Selection 3 (RNK-S3). Eighty eight to 93% of E+GA treated minitubers from cultivars in the long dormancy group developed sprouts signaling the end of dormancy within 5 weeks, while the control and ABA treated minitubers were totally dormant with 0% bud break. With the intermediate cultivars E+GA treated minitubers had 100% bud break compared to 37 to 55% bud break in the control minitubers. Treated long dormant minitubers also had 2.1 to 2.5 more sprouts per minituber and 1.6 to 2.4 cm longer sprouts. E+GA treated minitubers planted in field plot trials produced mature plants that were 19 to 36 cm per plant taller, with up to 3.9 to 8.8 more seed tubers per plant, and 1.9 to 4.2 kg per plot higher yields. The laboratory protocol developed to predict dormancy breaking attributes provided a reasonable estimate of cultivar dormancy under field conditions.
DOI:10.1111/j.1399-3054.2006.00752.xURL [本文引用: 1]
Fruit ripening is characterized by progressive depolymerisation of pectic and hemicellulosic polysaccharides of the cell wall. Several cell-wall hydrolases have been studied and their role implicated in the softening of various fruits. However, studies related to pectate lyases (PL), which degrade pectins through -elimination of glycosidic linkages, have been very limited in fruit tissue. We have cloned a PL homologue, MiPel1 from ripening mango ( Mangifera indica var. Dashehari), which shows sequence similarity to higher plant PL genes. The 1.7-kb complementary DNA of MiPel1 (accession number AY987389) encodes a putative protein of 437 amino acids, which possesses all the motifs and active site residues conserved in other PL. A progressive increase in transcript accumulation was observed during ripening but expression was delayed significantly in 1-methylcyclopropene-treated and control fruits kept in air without exogenous ethylene. The expression was specific to fruits and triggered only during ripening. No transcript was detected in other tissues such as leaf, stem and flower and during development of the fruit. The increase in transcript accumulation of MiPel1 during ripening was associated with an increase in the Ca +2 -dependent PL activity and pectin solubilization. It is proposed that expression of PL may be closely associated with pectin degradation during ripening and play an important role in mango softening.
DOI:10.1016/j.postharvbio.2010.12.014URL [本文引用: 1]
Petal abscission requires extensive dissolution of the middle lamella which is rich in pectins and pectic polysaccharides. The action of some wall hydrolases in abscission has been shown but the role of many other genes such as pectate lyase is still not clear. We have isolated a pectate lyase gene, Research highlights? We have isolated a pectate lyase gene, RbPel1, that is active during petal abscission. ? RbPel1 transcripts accumulate during both, ethylene induced and field abscission. ? No major change in expression is observed in non-abscising flowers of Rosa hybrida. ? RbPel1 action may be important for dissolution of middle lamella during abscission.
DOI:10.1104/pp.15.01556URLPMID:26829982 [本文引用: 1]
Abstract Leu-Leu-Met (LLM)-domain B-GATAs are a subfamily of the 30-membered GATA transcription factor family from Arabidopsis. Only two of the six Arabidopsis LLM-domain B-GATAs, i.e. GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED (GNC) and its paralog GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 (GNL), have already been analyzed with regard to their biological function. Together, GNC and GNL control germination, greening, flowering time, and senescence downstream from auxin, cytokinin (CK), gibberellin (GA), and light signaling. Whereas overexpression and complementation analyses suggest a redundant biochemical function between GNC and GNL, nothing is known about the biological role of the four other LLM-domain B-GATAs, GATA15, GATA16, GATA17, and GATA17L (GATA17-LIKE), based on loss-of-function mutant phenotypes. Here, we examine insertion mutants of the six Arabidopsis B-GATA genes and reveal the role of these genes in the control of greening, hypocotyl elongation, phyllotaxy, floral organ initiation, accessory meristem formation, flowering time, and senescence. Several of these phenotypes had previously not been described for the gnc and gnl mutants or were enhanced in the more complex mutants when compared to gnc gnl mutants. Some of the respective responses may be mediated by CK signaling, which activates the expression of all six GATA genes. CK-induced gene expression is partially compromised in LLM-domain B-GATA mutants, suggesting that B-GATA genes play a role in CK responses. We furthermore provide evidence for a transcriptional cross regulation between these GATAs that may, in at least some cases, be at the basis of their apparent functional redundancy. 2016 American Society of Plant Biologists. All Rights Reserved.
DOI:10.1016/j.devcel.2010.10.023URLPMID:3022420 [本文引用: 1]
Light and brassinosteroid (BR) antagonistically regulate the developmental switch from etiolation in the dark to photomorphogenesis in the light in plants. Here, we identify GATA2 as a key transcriptional regulator that mediates the crosstalk between BR- and light-signaling pathways. Overexpression of GATA2 causes constitutive photomorphogenesis in the dark, whereas suppression of GATA2 reduces photomorphogenesis caused by light, BR deficiency, or the constitutive photomorphogenesis mutant . Genome profiling and chromatin immunoprecipitation experiments show that GATA2 directly regulates genes that respond to both light and BR. BR represses Graphical AbstractView high quality image (189K)
DOI:10.5511/plantbiotechnology.17.0727aURL [本文引用: 1]
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DOI:10.1007/s10059-013-2270-4URLPMID:23314608 [本文引用: 1]
The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.
DOI:10.1111/tpj.12228URLPMID:23647236 [本文引用: 1]
The ICK/KRP cyclin-dependent kinase (CDK) inhibitors are important plant cell cycle factors sharing only limited similarity with the metazoan CIP/KIP family of CDK inhibitors. Little is known about the specific functions of different ICK/KRP genes in planta. In this study, we created double and multiple mutants from five single Arabidopsis ICK/KRP T-DNA mutants, and used a set of 20 lines for the functional investigation of the important gene family. There were gradual increases in CDK activity from single to multiple mutants, indicating that ICK/KRPs act as CDK inhibitors under normal physiological conditions in plants. Whereas lower-order mutants showed no morphological phenotypes, the ick1ick2ick6ick7 and ick1ick2ick5ick6ick7 mutants had a slightly altered leaf shape. The quintuple mutant had larger cotyledons, leaves, petals and seeds than the wild-type control. At the cellular level, the ICK/KRP mutants had more but smaller cells in all the organs examined. These phenotypic effects became more apparent as more ICK/KRPs were downregulated, suggesting that to a large extent ICK/KRPs function in plants redundantly in a dosage-dependent manner. Analyses also revealed increased expression of E2F-dependent genes, and elevated RBR1 as well as an increased level of phospho-RBB1 protein in the quintuple mutant. Thus, downregulation of multiple ICK/KRP genes increases CDK activity, upregulates the E2F pathway and stimulates cell proliferation, resulting in increased cell numbers, and larger organs and seeds.
DOI:10.1007/BF00192794URLPMID:24178317 [本文引用: 1]
The defense-related STH—2 gene is rapidly activated following infection or elicitor treatment of potato (Solanum tuberosum L.) tubers. However, its physiological or biochemical function is unknown. To study the STH—2 gene product and its accumulation during the defense response, we raised antibodies to a β-galactosidase-STH—2 fusion protein in Escherichia coli. The antiserum specifically recognized a protein of the predicted 17-kDa size in extracts of elicited tuber disks when analyzed by Western blot. In control extracts this band was not detected. The accumulation of STH—2 protein in response to incompatible and compatible zoospores of Phytophthora infestans (Mont.) de Bary depended on the inoculum density applied. Whereas a low concentration of spores induced accumulation of STH—2 protein faster in the incompatible than the compatible interaction, this difference in timing was less pronounced at higher inoculum densities. Inoculation with a high concentration of compatible spores also resulted in the disappearance of STH—2 protein late during the infection. In both control and induced tuber tissue the antibody strongly reacted with an unknown protein of 18 kDa. This protein was present constitutively in tubers, but in leaves its accumulation was stimulated by inoculation with P. infestans.
DOI:10.1007/BF00027364URLPMID:8358029 [本文引用: 1]
Abstract The STH-2 gene is rapidly activated in potato leaves and tubers following elicitation or infection by Phytophthora infestans. However, its biochemical function remains unknown. In order to ascertain if STH-2 protein is directly involved in the defense of potato against pathogens, the STH-2 coding sequence under the control of the CaMV 35S promoter was introduced into potato plants. Transgenic plants expressing the STH-2 gene were analyzed for an altered pattern of susceptibility to a compatible race of P. infestans and to potato virus X. Results indicate that constitutive expression of the STH-2 gene did not reduce susceptibility of potato to these pathogens.
DOI:10.1016/j.jbiotec.2010.09.934URLPMID:20854851 [本文引用: 1]
The PR-10a protein (formerly STH-2) is known to be induced by biotic stress in potato. The present study demonstrates that transgenic suspension cells of the potato cultivar Desiree over-expressing the PR-10a protein exhibit significantly increased salt and osmotic tolerance compared to the respective wild type cells. A comparison of the proteome pattern of Solanum tuberosum suspension cultures cv. Desiree before and after the treatment with NaCl or sorbitol under equiosmolar conditions (740 mOs/kg) revealed the pathogenesis related protein PR-10a to be one of the predominant differentially expressed proteins in potato cell cultures. The pr-10a mRNA was confirmed to be present by RT-PCR from salt challenged suspension cells and was transcribed into cDNA. For PR-10a over-expression Agrobacterium tumefaciens mediated transformation of the potato cells and a dicistronic vector harboring the cDNA of the pr-10a gene linked to a luciferase gene by an IRES (Internal Ribosome Binding Site) was used. The IRES mediated translation leads to co-expression of PR-10a and luciferase in a fixed ratio. By non-invasive luciferase assay homologous PR-10a over-expressing callus was identified after selection on phosphinothricin supplemented medium. This callus was used for the setup of a transgenic suspension culture. Along with increased salt and osmotic tolerance the transformed culture showed changed proline and glutathione levels under abiotic stress conditions in comparison to the wild type.
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