DataSheet_1_Ticarcillin degradation product thiophene acetic acid is a novel auxin analog that promotes organogenesis in tomato.pdf

Efficient regeneration of transgenic plants from explants after transformation is one of the crucial steps in developing genetically modified plants with desirable traits. Identification of novel plant growth regulators and developmental regulators will assist to enhance organogenesis in culture. In this study, we observed enhanced shoot regeneration from tomato cotyledon explants in culture media containing timentin, an antibiotic frequently used to prevent Agrobacterium overgrowth after transformation. Comparative transcriptome analysis of explants grown in the presence and absence of timentin revealed several genes previously reported to play important roles in plant growth and development, including Auxin Response Factors (ARFs), GRF Interacting Factors (GIFs), Flowering Locus T (SP5G), Small auxin up-regulated RNAs (SAUR) etc. Some of the differentially expressed genes were validated by quantitative real-time PCR. We showed that ticarcillin, the main component of timentin, degrades into thiophene acetic acid (TAA) over time. TAA was detected in plant tissue grown in media containing timentin. Our results showed that TAA is indeed a plant growth regulator that promotes root organogenesis from tomato cotyledons in a manner similar to the well-known auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). In combination with the cytokinin 6-benzylaminopurine (BAP), TAA was shown to promote shoot organogenesis from tomato cotyledon in a concentration-dependent manner. To the best of our knowledge, the present study reports for the first time demonstrating the function of TAA as a growth regulator in a plant species. Our work will pave the way for future studies involving different combinations of TAA with other plant hormones which may play an important role in in vitro organogenesis of recalcitrant species. Moreover, the differentially expressed genes and long noncoding RNAs identified in our transcriptome studies may serve as contender genes for studying molecular mechanisms of shoot organogenesis.

转化后从外植体高效再生转基因植株，是培育具备优良性状的转基因植物的关键环节之一。筛选新型植物生长调节剂与发育调控因子，有助于提升离体培养中的器官发生效率。本研究中，我们观察到在添加替门汀（timentin）的培养基中培养的番茄子叶外植体，其芽再生效率显著提升；替门汀是一类常用于抑制农杆菌（Agrobacterium）转化后过度增殖的抗生素。对添加与未添加替门汀的外植体开展比较转录组分析，我们鉴定出多个此前已被报道在植物生长发育中发挥重要作用的基因，包括生长素响应因子（Auxin Response Factors, ARFs）、GRF互作因子（GRF Interacting Factors, GIFs）、开花位点T（Flowering Locus T, SP5G）、生长素上调小RNA（Small auxin up-regulated RNAs, SAUR）等。部分差异表达基因通过实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）完成了验证。我们证实，替门汀的主要成分替卡西林（ticarcillin）可随时间降解为噻吩乙酸（thiophene acetic acid, TAA）。在添加替门汀的培养基中培养的植物组织内，可检测到TAA的存在。研究结果表明，TAA确实是一种植物生长调节剂，可通过与经典生长素吲哚-3-乙酸（indole-3-acetic acid, IAA）、吲哚-3-丁酸（indole-3-butyric acid, IBA）相似的作用方式，促进番茄子叶的根器官发生。当与细胞分裂素类物质6-苄氨基腺嘌呤（6-benzylaminopurine, BAP）联用时，TAA可呈浓度依赖性地促进番茄子叶的芽器官发生。据我们所知，本研究首次证实了TAA在植物物种中作为生长调节剂的功能。本研究将为后续探索TAA与其他植物激素的不同组合方案奠定基础，这些组合有望在顽拗型物种的离体器官发生研究中发挥重要作用。此外，本转录组研究中鉴定出的差异表达基因与长链非编码RNA（long noncoding RNAs），可作为研究芽器官发生分子机制的候选基因。